National Cancer Institute CARCINOGENESIS Technical Report Series No. 2 February 1976

CARCINOGENESIS BIOASSAY OF TRICHLOROETHYLENE

CAS No. 79-01-6

NCI-CG-TR-2

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health

NCI-CG-TR-2

February 1976

CARCINOGENESIS BIOASSAY OF TRICHLOROETHYLENE

CAS No. 79-01-6

These studies were conducted by Hazleton Laboratories, Inc., initially under direct contract to the National Cancer Institute and subsequently under subcontract with Tracor Jitco, Inc., Prime Contractor for the Carcinogenesis Bioassay Program, National Cancer Institute.

CARCINOGEN BIOASSAY AND PROGRAM RESOURCES BRANCH CARCINOGENESIS PROGRAM DIVISION OF CANCER CAUSE AND PREVENTION NATIONAL CANCER INSTITUTE BETHESDA, MARYLAND

DHEW Publication No. (NIH) 76-802

For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. 20402 - Price $3.30 Stock Number 017-042-00117-0

FOREWORD

One of the major goals of the National Cancer Institute is to determine the causative factors responsible for human cancer as a basis for preventive measures, both at the environmental and at the host levels. The identification of chemical and physical agents which represent carcinogenic hazards has been recognized as an essential task. The Carcinogenesis Program of the Division of Cancer Cause and Prevention includes among its responsibilities that of testing chemicals for carcinogenic activity.

Methods for carcinogenesis bioassay have continued to evolve in the last few decades. While progress was initially slow, and bioassay methods were crude, methodology has greatly improved within the past decade. With better bioassay testing procedures and more extensive efforts in this direction, more chemicals capable of causing cancer in humans will be detected first through experimental tests rather than exclusively through epidemiological studies. More than ever the experimentalists and epidemiologists can now interact to provide direction and leads to the enormous task of exploring the complex problems associated with chemicals and cancer in our society.

Technological developments of the last few decades have resulted in thousands of chemicals being introduced into the environment. A number of these chemicals can be expected to be found carcinogenic. The number of chemicals which have been shown to have carcinogenic activity has continued to increase.

Two of the major goals of the Carcinogenesis Program are to identify carcinogenic chemicals and to develop improved methodology for testing. The most reliable test method avail­ able at this time is the long term bioassay study using labora­ tory rodents. This is the primary procedure used currently by the bioassay program in its systematic testing for carcino­ genicity.

Due to the lack of in-house facilities adequate for conducting carcinogenesis bioassay studies, the NCI Carcinogenesis Program has implemented this activity through collaborative research contracts.

Several hundred chemicals have been selected for bioassay in recent years. Included among these is a series of chlorinated organic compounds. The bioassay of trichloroethylene, the subject of the present report, is one of the first of this series to have been completed.

The selection of this test dates back to decisions made in the early phases of implementation of the Carcinogenesis Program during the development of research on screening methods for carcinogenicity testing. Trichloroethylene was one of 18 chemicals tested under a contract awarded to Hazleton Labora­ tories, Incorporated. Vienna, Virginia, on May 1, 1971, as a result of a Request for Proposals advertised in the C£mmerc_e o n March 15, 1969.

This bioassay was initiated in 1972 according to the methods used and widely accepted at that time; it represents a valid carcinogenesis test. The design of carcinogenesis bioassays has evolved since then in some respects and several improvements have been developed. The currently recommended procedures are described in detail in the first volume of this series (NCI-CG­ TR-1) entitled "Guideline for Carcinogenesis Testing in Small Rodents" (1976). The most notable changes pertain to pre­ liminary toxicity studies, number of controls used, and extent of pathological examination.

The present report, the first of its kind, provides a detailed documentation of all the aspects of the bioassay, including all the individual animal data and diagnoses.

The publication of such detailed reports fulfills a commitment made in 1968 when NCI developed its ''Plan for Chemical Carcino­ genesis and the Prevention of Cancers". Methods and capabili­ ties for a fully detailed documentation and publication of well defined and relatively large bioassay studies had to be developed by the Program. They include the development of the Carcinogenesis Bioassay Data System, guidelines for bioassay protocols and procedures, and a network of bioassay resources for chemicals, animals, testing facilities, experimental design, pathology, data processing, and statistical analysis. Methods and criteria for pathological diagnosis and classification were developed.

This type of publication also fulfills a recommendation made by the International Union Against Cancer (UICC) in 1969 (Beren­ blum, 1969). In fact, the definition and exhaustive documenta­ tion of carcinogenesis bioassays was recommended by the UICC international expert panels. Those recommendations served as an essential basis for the development of the present reporting and publication system. A workshop on "Data Dissemination in Carcinogenesis" held by the NCI Carcinogenesis Program in January 19 74 endorsed the Technical Report serie^s as a means for disseminating carcinogenesis test results.

The present bioassay is clearly the result of team effort, Many people contributed to the selection of the test, the

ii development and design of the protocols and the diagnostic and analytical procedures, the establishment and monitoring of facilities, the conduct of the animal tests, and the analysis of results. All of them share in the authorship of this study. Their names and credit for their contribution to the study are given under the heading of "Contributors".

The interpretation of carcinogenesis bioassay results. in relation to the complex task of assessing human hazards, is beyond the scope of the present study. This report is designed to provide a factual basis for the interpretative efforts by giving a full, open, and detailed account of the observations made during this bioassay.

Norbert P. Page D.V.M Chief, Carcinogen Bioassay Umberto Saffiotti, M.D. and Program Resources Branch Associate Director, Carcinogenesis Program Carcinogenesis Program Division of Cancer Cause Division of Cancer Cause and Prevention and Prevention National Cancer Institute National Cancer Institute

111 CONTRIBUTORS

Many individuals and institutions have participated in the planning and conduct of this study. The selection of the chemical and test animals, design of the experiments, and much of the monitoring of progress was the responsibility of Dr. John Weisburger, previously of the National Cancer Institute, now with the American Health Foundation, and Dr. Elizabeth Weisburger, National Cancer Institute (NCI). Dr. John Weis­ burger served as project officer from inception of the contract until the fall of 1972. Drs. Elizabeth Weisburger and Norbert Page served in that capacity from that time until the present.

The actual animal experiments were conducted under contract to the Hazleton Laboratories, Incorporated (HLI), Vienna, Vir­ ginia. Principal Investigators for HLI were Drs. Willard Weath­ erholtz, William Olson, Marcelina Powers, and Richard Voelker. Dr. Robert Habermann conducted the microscopic examination of the tissues. Ms. Klara Petrovics was responsible for much of the routine technical aspects of the study. Tracor Jitco, Incorporated, Rockville, Maryland, as Prime Contractor for Bioassay Operations, with the assistance of the Hazleton Labo­ ratories, Incorporated and National Cancer Institute staffs, has prepared this report. Dr. Jane Robens was responsible for the coordination and major effort required in its preparation. Dr. Charlie Barron conducted a review and confirmation of the histopathologic diagnoses as submitted by Hazleton Laborato­ ries. Dr. Miles Davis conducted the statistical analysis, Dr. Stephen Olin prepared the chemical sections of this report, and Ms. Nancy Palmer functioned as Technical Editor.

The Biomedical Information Sciences Department,, EG&G/Mason Research Institute, Bethesda, Maryland, operations contractor for the Carcinogenesis Bioassay Data System (CBDS), under the direction of Mr. Dalton Tidwell, was responsible for the compi­ lation of the individual animal pathology tables and some of the summary tables. Drs. Norbert Page, Cipriano Cueto, and Umberto Saffiotti of the National Cancer Institute outlined the format of this report, worked closely with the Tracor Jitco and Hazleton Laboratories staffs in preparing it, reviewed its con­ tent, and contributed to the discussion and interpretation of the findings. Dr. John Gart, Head, Mathematics and Statistics Section, Field Studies and Statistics Program, Division of Cancer Cause and Prevention, NCI, and his staff wc»re responsi­ ble for verifying the accuracy of the data, tables, and the statistical analysis of the data.

iv Advisory Groups to the Carcinogenesis Program have provided guidance in the further development of the carcinogenesis bioassay methodology and the review of the Carcinogenesis Bioassay Program and its contracts. Members include Dr. Richard Adamson, NCI; Dr. Clyde Dawe, NCI; Dr. William Deichmann, University of Miami; Dr. Leo Friedman (deceased), Food and Drug Administration; Dr. John Gilbert, Harvard Computing Center; Dr. Harold Grice, Canadian Food and Drug Directorate; Dr. Paul Harris, Indianapolis, Indiana; Dr. Charles Irving, Memphis Veterans' Administration Hospital; Dr. Gerhard Krueger, NCI; Dr. Bernard McNamara, Edgewood Arsenal; Dr. Paul Newberne, Massachusetts Institute of Technology; Dr. Norbert Page, NCI; Dr. Lionel Poirier, NCI; Dr. William Priester, NCI; Dr. James Sontag, NCI; Dr. Robert Squire, NCI; Dr. Elizabeth Weisburger, NCI; Dr. John Weisburger, American Health Foundation; Dr. Harry Wood, NCI; and Mr. Samuel Poiley, NCI.

We are especially grateful for the contributions and valuable constructive criticism provided by the reviewers of this report. NCI staff reviewers were Drs. Thomas Cameron, John Cooper, Kenneth Chu, Cipriano Cueto, Herman Kraybill, Umberto Saffiotti, Sidney Siegel, James Sontag, Robert Squire, and Elizabeth Weisburger. Consultants who reviewed the report and provided valuable advice were Dr. Norman Breslow, University of Washington; Dr. Herbert Blumenthal, Food and Drug Administra­ tion; Dr. William D'Aguanno, Food and Drug Administration; Dr. Harold Grice, Canadian Food and Drug Directorate; Dr. Elton Homan, U.S. Environmental Protection Agency; Dr. Philip Issen­ berg, University of Nebraska Medical Center; Dr. William Lloyd, National Institute for Occupational Safety and Health; Dr. Roscoe Moore, National Institute for Occupational Safety and Health; Dr. Verald Rowe, Dow Chemical Company; Mr. Sheldon Samuels, American Federation of Labor/Congress of Industrial Organizations; Dr. Raymond Shapiro, Food and Drug Administra­ tion; Dr. Zeb Bell, Jr., PPG Industries, Inc.; Mr. Larry Sargert, PPG Industries, Inc.; Dr. John Weisburger, American Health Foundation; Dr. Jerome Wesolowski, California Department of Health.

In addition to those mentioned, appreciation is given to the numerous other staff personnel of Tracor Jitco, Inc., the National Cancer Institute's contractors, and the NCI Carcino­ genesis Program for their contributions to these studies.

While this Technical Report documents in detail the design, conduct, and results of the study, any further inquiries regarding the study should be directed to the Carcinogenesis Program of the National Cancer Institute.

SUMMARY

Trichloroethylene (TCE), a halogenated chemical, has been tested for carcinogenicity in the National Cancer Institute's Carcinogenesis Bioassay Program. Trichloroethylene has been used primarily as a solvent in industrial degreasing opera­ tions. Other uses have been as a solvent in dry cleaning and food processing, as an ingredient in printing inks, paints, etc., and as a general anesthetic or analgesic.

Industrial grade (>99% pure) trichloroethylene was tested using 50 animals per group at 2 doses and with both sexes of Osborne- Mendel rats and B6C3F1 mice. Twenty of each sex and species were maintained as matched controls, in addition to colony and positive carcinogen controls. Animals were exposed to the com­ pound by oral gavage 5 times per week for 78 weeks. At the end -of treatment, animals were observed until terminal sacrifice at 110 weeks for rats and 90 weeks for mice. A complete necropsy and microscopic evaluation of all animals (except 7 of the original 480) was conducted.

Two doses were used with animals started on test at approxi­ mately 6 weeks of age. The initial doses used in this test were the estimated maximum tolerated dose (MTD) and 1/2 MTD, as predicted from data obtained in a 6-week toxicity study. For rats, the initial doses were 1300 and 650 mg/kg body weight. These were changed, based upon survival and body weight data, so that the "time-weighted average" doses were 549 and 1097 mg/kg for both male and female rats. For mice, the initial doses were 1000 and 2000 mg/kg for males and 700 and 1400 mg/kg for females. The doses were increased so that the "time­ weighted average" doses were 1169 and 2339 mg/kg for male mice and 869 and 1739 mg/kg for female mice.

Clinical signs of toxicity, including reduction in weight, were evident in treated rats. These, along with an increased mortal­ ity rate, necessitated a reduction in doses during the test. In contrast, very little evidence of toxicity was seen in mice, so doses were increased slightly during the study. The increased mortality in treated male mice appears related to the presence of liver tumors.

A variety of neoplastic lesions were observed in rats with no significant difference between trichloroethylene-treated and control animals. The only lesion that might be attributed to

vii the treatment was a chronic nephropathy found in both sexes and at both dose levels.

With both male and female mice, primary malignant tumors of the liver, i.£.» hepatocellular carcinoma, were observed in high numbers. For males, 26/50 low dose and 31/48 high dose animals had hepatocellular carcinomas as compared with 1/20 matched controls and bill colony controls. The differences between treated and matched control males at both doses were highly significant (P < 0.01). For females, hepatocellular carcinomas were observed in 4/50 low dose and Wlkl high dose animals as compared with 0/20 matched controls and 1/80 colony controls. While the difference between the high dose female mice and matched controls was also highly significant (P < 0.01), the difference at the low dose was less (P = 0.09). For both male and female mice, age-adjusted tests for linear trend (dose response) were highly significant for hepatocellular carcinoma (P < 0.001 for males and P = 0.002 for females).

In male mice at the high doses, hepatocellular carcinomas were observed early in the study. The first was seen at 27 weeks; 9 others were found in male mice dying by the 78th week. The tumor was not observed so early in low dose male or female mice. The diagnosis of hepatocellular carcinoma was based on size, histologic appearance, and presence of metastasis, especially to the lung. No other lesion was significantly elevated (P < 0.05) in treated mice. The incidence of hepato­ cellular carcinomas in the trichloroethylene-matched controls was typical of that observed in colony controls.

Carbon tetrachloride (CCI4) was used as a positive control for the series of chlorinated chemicals which included trichloro­ ethylene. While virtually all male and female mice developed hepatocellular carcinomas following carbon tetrachloride treat­ ment, the response in the Osborne-Mendel rat was considerably less. Only about 5% developed hepatocellular carcinomas. Thus, there appears to be a marked difference in sensitivity to induction of carcinomas by chlorinated compounds between the B6C3F1 mouse and the Osborne-Mendel rat.

The results of this carcinogenesis test of trichloroethylene clearly indicate that trichloroethylene induced a hepato­ cellular carcinoma response in mice. While the absence of a similar effect in rats appears most likely attributable to a difference in sensitivity between the Osborne-Mendel rat and the B6C3F1 mouse, the early mortality of rats due to toxicity must also be considered.

viii TABLE OF CONTENTS

Page

1.0 INTRODUCTION 1

2 .0 MATERIALS 2

2.1 Name and Synonyms 2 2.2 Formula, Molecular Weight, Identifying Numbers, and Characteristics 2 2 .3 Procurement 2 2.4 Chemical Analysis 3 2.5 Preparations Used for All Bioassays 3 2.6 Safety Procedures 3

3 .0 TEST ANIMALS AND ENVIRONMENT 5

4.0 PRECHRONIC PHASES: METHODS AND RESULTS 7

4.1 Acute Study 7 4.2 Eight-Week Subchronic Study 7 4.2.1 Methodology 7 4.2.2 Results - Rats 8 4.2.3 Results - Mice 8 4.2.4 Selection of MTD 8

5.0 CHRONIC TESTING: METHODOLOGY 9

5 .1 Experimental Design 9 5.1.1 Experimental Groups 9 5.1.2 Dates of Study 9 5.1.3 Preparations and Doses 9 5.1.4 Treatment Schedule 10 5 . 2 Observations 10 5.3 Necropsy 10 5.4 Histological Preparation and Microscopic Examination.. 13 5.5 Data Processing and Confirmation 14 5.6 Positive Controls 15

6.0 CHRONIC TESTING: RESULTS - RATS 16

6.1 Body Weights 16 6.2 Clinical Observations 16 6 .3 Survival 16 6.4 Pathology 17 6.5 Tumor Probabilities 21 6.6 Controls 22 6.6.1 Survival 22 6.6.2 Tumors 22

IX 7 .0 CHRONIC TESTING: RESULTS - MICE. . . 24

7 .1 Body Weights 24 7 .2 Clinical Observations 24 7.3 Survival 24 7.4 Pathology 27 7.5 Tumor Probabilities 34 7 .6 Colony Controls 35 7.7 Positive Controls 39

8.0 DISCUSSION 41

8.1 Design of Bioassay 41 8.1.1 Selection of Animal Species 41 8.1.2 Route of Exposure 41 8.1.3 Use of High Doses 42 8.1.4 Methodology 42 8.2 Test Compound Purity 43 8.3 Metabolism, Distribution, and Excretion 43 8 .4 Toxicology 44 8.5 Pathology and Survival 45 8.5.1 Rats 45 8.5.2 Mice 45 8.6 Effect of Various Compounds in the Same Room 46 8.7 Relationship to Toxicity of Carbon Tetrachloride...... 46 8 .8 Conclusions 47

9 .0 BIBLIOGRAPHY 48

APPENDIX A: CHEMISTRY 53 Chemical and Physical Characteristics 54 Technical Product and Impurities 54 Manufacturing Processes 54 Chemical Analysis 55 Identification of Trace Components 61 Feed Analysis 66 Water Analysis 68

APPENDIX B: WEIGHTS AND SURVIVAL 69 Mean Body Weights and Survival - Subchronic Study 70 Mean Body Weights and Survival - Chronic Study 74

APPENDIX C: STATISTICS 87 Statistical Methodology 88 Data for Statistical Analysis 93 Survival Probabilities 97 Tumor Probabilities 102 APPENDIX D: PATHOLOGY 117 Tissues Examined - Trichloroethylene-Treated Animals 118 Tumor Summary Tables by Site of Origin and by Anatomic Site - Trichloroethylene-Treated Animals 120 Individual Pathology - Trichloroethylene-Treated Animals... 134

APPENDIX E: POSITIVE CONTROLS Individual Liver Pathology - Positive Control (Carbon Tetrachloride) Animals 17 7

XI

LIST OF TABLES

Page

Table I. Identification of Trichloroethylene Used in Study 2

Table II. Design and Survival Results - Trichloroethylene Subchronic Study 7

Table III. Dosage and Observation Schedule - Trichloroethylene Chronic Study 11

Table IV. Dosage and Observation Schedule ­ Carbon Tetrachloride Study 15

Table V. Tumor Incidence - Rats with Tumors 17

Table VI. Comparison of Survival of Colony Controls and Trichloroethylene- and Carbon Tetrachloride- Treated Rats 22

Table VII. Incidence of Liver Tumors - Colony Control and Carbon Tetrachloride-Treated Rats 22

Table VIII. Incidence of Hepatocellular Carcinoma ­ Trichloroethylene-Treated Mice 35

Table IX. Probability of Observing Hepatocellular Carcinoma in Trichloroethylene-Treated Male Mice 36

Table X. Incidence of Hepatocellular Carcinoma ­ Colony Control Mice 39

Table XI. Comparison of Survival in Colony Controls ­ Vehicle-Treated and Trichloroethylene­ and Carbon Tetrachloride-Treated Mice 39

Table Xlla. Comparison of Hepatocellular Carcinoma Incidence in Colony Controls - Vehicle Treated and Trichloroethylene- and Carbon Tetrachloride- Treated Mice 40

Table Xllb. Comparison of Time to Liver Tumor Detection in Colony Controls - Vehicle Treated and Trichloroethylene- and Carbon Tetrachloride- Treated Mice 40

Table Xllla. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Male Rats.... 70 Table Xlllb. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Female Rats., 71

xiii Table XlVa. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Male Mice.... 72

Table XlVb. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Female Mice.. 73

Table XVa. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Male Rats 74

Table XVb. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Female Rats 76

Table XVIa. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Male Mice 78

Table XVIb. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Female Mice 80

Table XVII. Identity of Tumor Marks . 92

Table XVIIIa. Data for Statistical Analysis ­ Trichloroethylene-Treated Male Rats 93

Table XVIIIb. Data for Statistical Analysis ­ Trichloroethylene-Treated Female Rats 94

Table XlXa. Data for Statistical Analysis ­ Trichloroethylene-Treated Male Mice . . 95

Table XlXb. Data for Statistical Analysis ­ Trichloroethylene-Treated Female Mice 96

Table XXa. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Male Rats.... 97

Table XXb. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Female Rats.. 98

Table XXc. Statistical Tests Comparing Estimated Probability of Survival among Control and Trichloroethylene-Treated Rats 99

Table XXIa. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Male Mice.... 100

Table XXIb. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Female Mice.. 100

xiv Table XXIc. Statistical Tests Comparing Estimated Probability of Survival among Control and Trichloroethylene-Treated Mice 101

Table XXII. Tumor Incidence - Trichloroethylene-Treated Rats.. 102

Table XXIIIa. Statistical Tests Comparing Estimated Probability of Observing Reticulum-cell Sarcoma, Lympho­ sarcoma, or Malignant Lymphoma (Mark b) among Control and Trichloroethylene-Treated Rats 103

Table XXIIIb. Statistical Tests Comparing Estimated Probability of Observing Fibroadenoma of the Mammary Glands (Mark g) among Control and Trichloroethylene- Treated Rats 104

Table XXIIIc. Statistical Tests Comparing Estimated Probability of Observing Hemangioma of Any Site (Mark h) among Control and Trichloroethylene-Treated Rats.. 104

Table XXIIId. Statistical Tests Comparing Estimated Probability of Observing Follicular Adenocarcinoma of the Thyroid (Mark p) among Control and Trichloro­ ethylene-Treated Rats 105

Table XXIIIe. Statistical Tests Comparing Estimated Probability of Observing Chromophobe Adenoma of the Pituitary (Mark t) among Control and Trichloroethylene- Treated Rats 105

Table XXIVa. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma of the Liver among Pooled Control and Trichloroethylene-Treated Rats. 106

Table XXIVb. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Trichloro­ ethylene-Treated Rats 107

Table XXIVc. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma of the Liver among Pooled Control and Carbon Tetrachloride- Treated Rats 108

Table XXIVd. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Carbon Tetrachloride-Treated Rats 109

xv Table XXV. Tumor Incidence - Trichloroethylene-Treated Mice.. Ill

Table XXVIa. Statistical Tests Comparing Estimated Probability of Observing Hepatocellular Carcinoma (Mark a) among Control and Trichloroethylene-Treated Mice.. 112

Table XXVIb. Statistical Tests Comparing Estimated Probability of Observing Reticulum-cell Sarcoma, Lympho­ sarcoma, or Malignant Lymphoma (Mark b) among Control and Trichloroethylene-Treated Mice 112

Table XXVIc. Statistical Tests Comparing Estimated Probability of Observing Carcinoma or Adenocarcinoma of the Lung or Alveoli (Mark c) among Control and Trichloroethylene-Treated Mice 113

Table XXVId. Statistical Tests Comparing Estimated Probability of Observing Adenoma of the. Lung (Mark d) among Control and Trichloroethylene-Treated Mice 113

Table XXVle. Statistical Tests Comparing Estimated Probability of Observing Carcinoma, Adenocarcinoma, or Adenoma of the Lung or Alveoli (Mark c or d) among Control and Trichloroethylene-Treated Mice.. 114

Table XXVII. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma among Pooled Control and Trichloroethylene-Treated Mice. 115

Table XXVIIIa. Numbers of Tissues Examined ­ Trichloroethylene-Treated Rats 118

Table XXVIIIb. Numbers of Tissues Examined ­ Trichloroethylene-Treated Mice 119

Table XXIXa. Primary Tumors by Site of Origin ­ Trichloroethylene-Treated Rats 121

Table XXIXb. Tumors by Anatomic Site ­ Trichloroethylene-Treated Rats 124

Table XXXa. Primary Tumors by Site of Origin ­ Trichloroethylene-Treated Mice 127

Table XXXb. Tumors by Anatomic Site ­ Trichloroethylene-Treated Mice 130

Table XXXIa. Individual Pathology - Trichloroethylene- Treated Male Rats 135

Table XXXIb. Individual Pathology - Trichloroethylene- Treated Female Rats 147

xvi Table XXXIla. Individual Pathology •- Trichloroethylene- Treated Male Mice 158

Table XXXIIb. Individual Pathology - Trichloroethylene- Treated Female Mice 168

Table XXXIIIa. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Rats 178

Table XXXIIIb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Rats 181

Table XXXIIIc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Rats 184

Table XXXIIId. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Rats.... 187

Table XXXIVa. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Mice 190

Table XXXIVb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Mice 192

Table XXXIVc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Mice 194

Table XXXIVd. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Mice.... 196

xvii LIST OF FIGURES

Page

Figure la. Dosage Schedule - Chronic Study ­ Trichloroethylene-Treated Rats 12

Figure lb. Dosage Schedule ­ Chronic Study ­ Trichloroethylene-Treated Mice 12

Figure 2a. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Male Rats.... 18

Figure 2b. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Female Rats.. 19

Figure 3a. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Male Mice.... 25

Figure 3b. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Female Mice.. 26

Figure 4. Primary hepatocellular carcinoma, mouse (high dose male #32). The rather well differentiated tumor of trabecular pattern has a recognizable boundary (—>) with the pre-existent hepatic tissue. Hematoxylin and eosin, x75 28

Figure 5 . Same as Figure 4, x300 28

Figure 6. Primary hepatocellular carcinoma, mouse (high dose male #26). The rather anaplastic solid tumor has a recognizable boundary (—>) with the pre-existent hepatic parenchyma. Mitoses are abundant. Hematoxylin and eosin, x240 30

Figure 7. Primary hepatocellular carcinoma, mouse (high dose male #15) with both solid trabecular pattern and papillary pattern. Most cells are large and resemble normal hepatocytes. There are also smaller cells with less cyto­ plasm and smaller, more basophilic nuclei. These cells usually occur in clusters and are especially prominent in the larger papillary structures 30

XVlll Figure 8. Primary hepatocellular carcinoma, mouse (high dose male #32). There is an area of highly anaplastic cells in a papillary pattern contiguous with an area of well differentiated cells in a trabecular pattern. Hematoxylin and eosin, xl20 32

Figure 9. Secondary hepatocellular carcinoma, lung, mouse (high dose male #15). The metastatic hepa­ tocellular carcinoma has invaded and extended into a bronchiole. The bulk of the tumor consists of rather well differentiated hepatocytes but there are scattered foci of smaller, more anaplastic, basophilic, neoplastic cells (—>) also. Hematoxylin and eosin, xl20 32

Figure 10. Metastatic hepatocellular carcinoma, lung, mouse (high dose male #15). Well differentiated hepato­ cytes comprise a large nodule (edge at upper left) and invade perivascularly at lower center. Numerous foci of smaller, more anaplastic, basophilic, neoplastic cells occur in vessels (—>) and alveolar capillaries, and invade perivascularly. Hematoxylin and eosin, x96 32

Figure 11. Product-Limit Estimates of Probability of Observing Hepatocellular Carcinoma ­ Trichloroethylene-Treated Male Mice 37

Figure 12. Comparison of Incidence of Hepatocellular Carcinoma in Trichloroethylene-Treated Male and Female Mice 38

Figure 13a. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Male Rats 82

Figure 13b. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Female Rats 83

Figure 14a. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Male Mice 84

Figure 14b. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Female Mice 85

xix

1.0 INTRODUCTION

In the late 1960s, scientists at the National Cancer Institute noted that a group of halogenated compounds extensively used as solvents in industrial processes had not been adequately tested for chronic toxicity. A related compound, carbon tetrachloride, however, had already been found carcino­ genic in mice (Eschenbrenner and Miller, 1944, 1946), hamsters (Delia Porta et al. , 1961), and rats (Reuber and Glover, 1970). Thus, carcinogenesis bioassays of a group of these solvents were initiated.

Trichloroethylene was one of the chemicals selected to be tested and this report describes the conduct and results of its bioassay. Production of trichloroethylene was reported as 609, 514, and 427 million pounds in 1970, 1971, and 1972, respectively, in the Chemical Economics Handbook (1972). The primary use (about 90%) of trichloroethylene is in the vapor degreasing of metals and equipment. It has also been used as a solvent in dry clean­ ing, in the processing of certain medicines and foods, and in other processes, as an ingredient in printing inks, paints, lacquers, varnishes, and adhesives, as a chemical intermediate, and in a variety of other applications such as a grain fumigant (Wiseman, 1972; Frear, 1969).

A pharmaceutical grade of trichloroethylene has also been used as a general anesthetic in surgical and obstetrical procedures, administered by inhala­ tion. It is a potent analgesic but will not produce appreciable skeletal muscle relaxation at the concentrations used. As an analgesic it has been used for minor procedures such as cleaning and debridement of burns, orthopedic manipulations, cystoscopy, incision of abscesses, surface biopsy, changing painful dressings, and treating trigeminal neuralgia (Price and Dripps, 1965).

Trichloroethylene has been identified in low concentrations in certain municipal water supplies as reported by the Environmental Protection Agency (Dowty et al., 1975). Residues may result from the use of trichloro­ ethylene as a solvent in the processing of foods. Tolerances for trichloroethylene of 25 ppm in decaffeinated ground coffee, 10 ppm in decaffeinated soluble (instant) coffee extract, and 30 ppm in spice oleoresins have been established by the Food and Drug Administration (Code of Federal Regulations, Title 21) . Thus, exposure may occur indirectly to the general population through residues in water and food.

The National Institute of Occupational Safety and Health (1973) has issued a comprehensive review of the uses, exposure, and known biological effects of trichloroethylene. Criteria for a recommended standard for occupational exposures are given in this document. It recommends that occupational exposure to trichloroethylene be controlled so that no worker shall be exposed to a peak concentration of trichloroethylene in excess of 150 ppm as measured over a maximum sampling time of 10 minutes, or to a concentra­ tion in excess of 100 ppm determined as a time-weighted average exposure for an 8-hour workday as measured over a minimum sampling time of 10 minutes. The Occupational Safety and Health Administration has recently proposed changes in their regulations to reflect these recommendations. 2.0 MATERIALS

2.1 Name and Synonyms

Chemical Abstracts and IUPAC Name: Trichloroethene Synonyms and Common Name: Trichloroethylene Acetylene trichloride Ethinyl trichloride 1,1,2-Trichloroethylene TCE (Christensen and Luginbyhl, 1974; Deichmann and Gerarde, 1969)

2.2 Formula, Molecular Weight, Identifying Numbers, and Characteristics

Formula: C2HCI3

Molecular Weight: 131.40 Wiswesser Line Notation: GYGU1G Chemical Abstracts Service Registration Number: 79-01-6 NCI Number: CO4546

For chemical and physical characteristics, technical product and impurities, and manufacturing processes, see Appendix A.

2.3 Procurement

Four batches of trichloroethylene were procured from Aldrich Chemical Company as given in Table I:

Table I. Identification of Trichloroethylene Used in Study

Batch Manufacturer's Received by Analysis Report No. Lot No. Hazleton (date) (date) Use

1 050191 7/23/71 1/9/73 Prechronic study and first 2 weeks of chronic study. 2 061891 7/23/71 Weeks 3-15 in chronic study. 3 063017 5/5/72 2/16/74 Weeks 16-36 in chronic study. 4 063014 10/12/72 7/8/74 Weeks 37-78 in chronic study. Each batch was received in one or more large amber bottles. Containers were stored in the dark at room temperature.

2.4 Chemical Analysis

The purity of the trichloroethylene used in the bioassay was determined by gas chromatography and infrared spectroscopy. Minor components subse­ quently were identified by gas chromatography-mass spectrometry and con­ firmed with reference standards.

Analyses of gas chromatographic total area data showed the major component to be at least 99% in each batch. Infrared spectra compare well with trichloroethylene reference spectra. The minor components comprise a mixture of stabilizers routinely added to commercial formulations of trichloroethylene. They include 1,2-epoxybutane (0.19%), ethyl acetate (0.04%), epichlorohydrin (0.09%), N-methylpyrrole (0.02%), and diisobutylene (0.03%) . Percentages were determined by FID gas chromatography with standards after completion of the bioassay. No detect­ able quantities of 1, 1, 2, 2-tetrachloroethane (<5 ppm) or 1,1,1,2-tetra­ chloroethane (<2 ppm) were indicated by gas chromatography, using reference standards. (For details of chemical analysis, jse_e Appendix A.)

2•5 Preparations Used for All Bioassays

Fresh solutions of trichloroethylene in corn oil were prepared weekly in amounts sufficient to treat all animals for one week, sealed, and refrigerated until use. Concentrations for the chronic test are given in Table III. The corn oil (purchased from the distributor, C. F. Sauer Company, Richmond, VA) was not analyzed for impurities or reaction products during this study.

2.4 Safety Procedures

Laboratory personnel working with undiluted experimental compound were required to wear the following protective gear: safety goggles, latex gloves, disposable full-body protective suit with attached feet and open-face hood, half-face Welsh respirator fitted with a dust and mist filter over a chemical cartridge for organic vapors. All work with these materials was conducted in a negative-pressure room and under a hood. Personnel involved with compound administration, animal weighing, and feeding wore a disposable laboratory coat, head covering, the Welsh respirator, and disposable latex gloves. Personnel involved with animal care, i-e., cage changing and washing, wore heavy duty gloves, safety shoes, half-face surgical mask (3M), and head covering. Any person enter­ ing the animal rooms was required to wear a head covering and 3M mask. As an additional personnel safety measure and to minimize cross contamination within animal rooms, actual intubation procedures were performed within the confines of a fume hood. Test solutions were kept in an ice bath during the dosing procedures to minimize evaporation. In the rooms housing rats, hoods were located in the corner of the room and each rack was wheeled to the hood each time the animals were dosed. Mouse racks were transported through the hall to another room with a large hood and each cage was placed directly under the hood during the intubation process. The testing laboratory's health and safety officer in conjunction with laboratory personnel made on-site inspections to insure compliance with the above safety precautions. 3.0 TEST ANIMALS AND ENVIRONMENT

Random-bred Osborne-Mendel rats (Battelle Memorial Institute, Columbus, OH) and B6C3F1 (C57BL/6 x C3H/He) hybrid mice (Charles River, Wilmington, MA) in the chronic study were obtained at 35 and 25 days of age, respectively, from suppliers under contract to NCI. Trichloroethylene-treated rats and their controls were born within 6 days of each other, with a median birth date of February 23, 19 72. Trichloroethylene-treated mice and their con­ trols were born within 6 days of each other, with a median birth date of July 17, 19 72. Upon arrival at the laboratory, all animals were isolated for at least 10 days. They were observed at arrival and weighed imme­ diately before being placed on study. Weight ranges of trichloroethylene­ treated animals and their controls in the chronic study were: male rats, 168-229 g; female rats, 130-170 g; male mice, 11-22 g; female mice, 11-18 g. Animals were randomly* assigned to treatment groups, so that initially the average weight in each group was approximately the same.

The rats were individually housed in hanging galvanized steel cages, 25.4 x 17.8 x 17.8 cm (Wahmann) with wire mesh fronts and floors. There were 72 cages per rack placed to allow 2 racks per 100 square feet of floor space. Paper collection trays (National Paper Products) were placed under the cages and were changed twice weekly. The rats were transferred to freshly cleaned cages weekly. The soiled cages and racks were washed under pressure at 80°C in water containing Super Soilax detergent (Economics Laboratory, Inc.), rinsed at 80°C, and steamed in a Matawan 375 gallon automatic cycle industrial washer. The water bottles with stainless steel sipper tubes inserted in rubber stoppers were changed twice weekly; the dirty bottles and tubes were washed, rinsed, and steamed in a 220 gallon industrial washer similar to that used for cage washing. Feed was supplied in glass jars within each cage. Trichloroethylene-treated rats and their controls were maintained in a room housing other rats being treated with one of the following compounds: dibromochloropropane, ethylene dichloride, 1,1- dichloroethane, and carbon disulfide. Four groups of vehicle-treated controls were in the same room.

The mice in the chronic phase were housed in polypropylene cages (Lab Products), 47 x 24.1 x 15.2 cm, which contained 10 animals of one sex per cage. (In the prechronic phases mice were housed individually in hanging wire mesh steel cages, 17.8 x 12.7 x 10.2 cm.) Animal rooms contained 40 cages per rack to allow 1.5 racks per 100 square feet of floor space. The cover for each unit was welded stainless steel wire over which was placed a non-woven polyester fiber filter bonnet. Each cage contained a galvanized iron, compartmentalized lfgangif feeder (Dixie Sheet Metal Co.) with a screen top. All mice were transferred to clean cages containing fresh bedding (Sanichips, a heat-treated hardwood product, Shurfire Products, Inc.) twice weekly. The soiled cages were washed and rinsed at 80°C and steamed. The steel wire bar covers of the cages were washed on a weekly basis and the racks on a monthly basis. The filter bonnets were washed and autoclaved weekly. The water bottles and stainless steel drinking tubes were changed

^•Animals were not distributed according to a table of random numbers.

5 3 times a week and cleaned by washing, rinsing, and steaming as stated ab o ve.

Mice treated with trichloroethylene were maintained in a room housing other mice being treated with one of the following 17 compounds: 1,1,2,2-tetra­ chloroethane, chloroform, 3-chloropropene, chloropicrin, 1,2-dibromochloro­ propane, 1,2-dibromoethane, ethylene dichloride, 1,1-dichloroethane, 3-sulfolene, iodoform, methyl chloroform, 1,1,2-trichloroethane, tetra­ chloroethylene, hexachloroethane, carbon disulfide, trichlorofluoromethane, and carbon tetrachloride. Nine groups of vehicle controls and 9 groups of untreated controls were also housed in this room.

All feeders for both rats and mice were changed weekly for the first 10 weeks and every 4 weeks thereafter and were cleaned by the same process of washing, rinsing, and steaming.

Following changing, the clean cages for all animals were placed on the racks in the same manner as before; however, the racks were repositioned within the room on a daily basis for the first 78 weeks, and weekly thereafter. The floors of each room were cleaned daily using Mikro-Bac, a phenolic detergent-disinfectant (Economics Laboratory, Inc.).

The total air in each room was changed 10-15 times per hour with all incoming air filtered through 2-inch thick fiberglass disposable filters which were changed at least once weekly. The relative humidity of the room air was maintained between 45 and 55% and the temperature range was 20 to 24°C. Rooms were illuminated by fluorescent lighting 12 hours per day. There was no communication between rooms, _i.j^.> there were no connecting doors, separate groups of technicians handled the rats and mice, each room had individual air ducts, and rooms were under negative pressure. Samples of ambient air were not tested for presence of volatile materials.

Wayne Lab-Blox meal was fed to the animals aid libitum. (Appendix A contains a feed ingredient list, analysis of protein, fat, and fiber by the manufacturer, and analyses for pesticide residues in selected feed batches.) Drinking water, from a local artesian well, was supplied ad libitum in glass bottles. (Appendix A presents a water analysis.)

Hazleton Laboratories, Inc., was certified as a research facility in August 1967 under the Animal Welfare Act by the USDA, Animal and Plant Health Inspection Service. The animal care facilities were fully accredited by the American Association for Accreditation of Laboratory Animal Care beginning June 4, 1971.

6 4.0 PRECHRONIC PHASES: METHODS AND RESULTS

4•1 Acute Study

Single-dose range-finding studies were conducted with male rats and female mice to determine the highest dose to be used in the 8-week subchronic study. Groups of 2 animals each were administered a single dose of trichloroethylene in corn oil by gavage by oral intubation and observed for 14 days. Ten dosages were used: 100, 178, 316, 562, 1000, 1420 (rats only), 1780 (mice only), 3160, 5620, 10,000, and 17,800 mg/kg. The lowest doses causing death, 5620 mg/kg for rats and 10,000 mg/kg for mice, were selected as the highest doses to be used in the 8-week subchronic study.

4.2 Eight-Week Subchronic Study

The objective of this study was to estimate the Maximum Tolerated Dose (MTD) for trichloroethylene to be used in rats and mice in the bioassay for carcinogenicity. In this context the MTD is defined as the highest dose that can be administered during the chronic study, which will not be expected to alter the animals' survival rate from effects other than carcinogenicity.

4.2.1 Methodology

Animals were placed into 6 groups each of 5 males and 5 females so that initially the average weight per animal in each treatment group was the same. Five groups received the test compound at varying dosages; one group served as the control and received only the vehicle (corn oil).

Trichloroethylene was dissolved in corn oil and animals were dosed by gavage under a hood for 5 consecutive days per week for 6 weeks on the basis of milligrams trichloroethylene per kilogram body weight. Doses ranged from 562 to 5620 mg/kg in rats and from 1000 to 10,000 mg/kg in mice (Table II).

Table II. Design and Survival Results - Trichloroethylene Subchronic Study

Rats Mice Group Dose Concn Survival0 Dose Concn Survival0 No. (mg/kg)a (mg/ml)b Males Females (mg_/kg)a (mg/ml)b Males Females

1 0 0 5/5 5/5 0 0 5/5 5/5 2 562 562 5/5 5/5 1000 100 5/5 5/5 3 1000 1000 5/5 5/5 1780 178 5/5 5/5 4 1780 1780 5/5 5/5 3160 316 5/5 3/5 5 3160 3160 5/5 5/5 5260 562 1/5 1/5 6 5620 5620 0/5 0/5 10,000 1000 0/5 0/5

amg Trichloroethylene/kg body weight, bmg Trichloroethylene/ml corn oil. cAt 8 weeks. Animals were weighed weekly and the most recent weight was used as a guide for the dosage. All animals of one sex within a treatment group received the same dosage, that is, the volume administered to all animals was based on the mean body weight for the group.

Dosing was stopped after 6 weeks and the animals were maintained for an additional 2 weeks under control conditions to detect delayed toxicity. Body weight was recorded on day 0 and weekly thereafter. Food consumption and observations of appearance, behavior, and signs of toxic effects were recorded weekly. Observations of mortality were made daily. Each animal that died and all animals killed at termination at 8 weeks were gross necropsied. No histopathology was performed. Mean body weights for each group, including standard deviations, and survival by week were determined (Tables XIIIa, Xlllb, XlVa, and XlVb, Appendix B) .

4.2.2 Results - Rats

While body weight gains of all treated groups were below those of controls, the reduction exceeded 20% for doses above 1780 for females and 3160 mg/kg/day for males.

No abnormal clinical signs were evident for doses of 1780 mg/kg/day and below. Hunching, discoloration of the fur due to urine stains, alopecia, and labored respiration were observed at 3160 and 5620 mg/kg/day. Kidney lesions were observed in 2 males at 1780 mg/kg/day, one a dilated kidney pelvis and the other a dark red renal medulla. Incidental findings included large abscessed areas in all lobes of the lungs of 2 test animals. No other gross lesions were noted.

4.2.3 Results - Mice

Body weight gains in all surviving groups were not significantly affected in a dose-related manner. Except for death at the higher doses, there were no signs attributable to the compound. All survivors appeared normal at termination. No lesion was noted in any mouse at necropsy.

4.2.4 Selection of MTD

Based on body weight gains and survival rates, the initial high doses (estimated maximum tolerated dose) were selected as 1300 mg/kg for both male and female rats, 2000 mg/kg for male mice, and 1400 mg/kg for female mice.

8 5.0 CHRONIC TESTING: METHODOLOGY

5.1 Experimental Design

5.1.1 Experimental Groups

Trichloroethylene was administered at 2 doses to both sexes of Osborn- Mendel rats and B6C3F1 mice, in groups of 50 animals each. Therefore, a total of 400 treated animals divided into 8 groups was used. Groups of 20 matched-*- vehicle-treated controls were used for each sex of each species. Ninety-nine male and 98 female rats and 77 male and 80 female mice were used as vehicle-treated "colony controls*', and 70 male and 76 female mice were used as untreated colony controls. They served as matched controls to trichloroethylene and to other compounds that were tested simultaneously. A group of positive control animals treated with carbon tetrachloride was also studied; see section 5.6. Treated and control animals came from the same source and were otherwise comparable. Animals were randomly^ assigned to treatment and control groups, so that initially the average weight in each group was approximately the same.

5.1.2 Dates of Study

Rats receiving trichloroethylene and their controls were placed on study at 48 days of age on April 11, 1972 and killed after 110 weeks on May 23, 19 74. Mice receiving trichloroethylene and their controls were placed on study at 35 days of age on August 21, 1972 and killed after 90 weeks on May 15, 1974.

5.1.3 Preparations and Doses

Trichloroethylene was dissolved in corn oil at concentrations of 60% w/v for rats and 10-24% w/v for mice and administered by gavage for 5 consecu­ tive days per week. The amount of solution to be administered was calcu­ lated weekly on the basis of the animal body weight, using the following factor: dose (mg/kg)/concentration (mg/ml) = F (ml/kg). For instance, a group of mice scheduled to receive trichloroethylene at a dose of 1000 mg/kg/day and weighing an average of 20 g at the end of week 2 actually received 0.13 ml of a 15% solution during week 3 (1000/15 = F = 6.6 ml/kg; 6.6 x 20/1000 = 0.13 ml/20 g) . Later in the study when weighing was done only once monthly, each newly calculated dose was administered for 4 weeks. All animals of one sex within a treatment group received the same dosage, that is, the volume of trichloroethylene solution administered to all animals was based on the mean body weight for the group. Controls received by gavage a dose of vehicle (corn oil) based on the factor calculated for the high dose group.

*• Vehicle-treated controls will be assumed to be matched, and will not be referred to as such in the remainder of this report. ^Animals were not distributed according to a table of random numbers. 5.1.4 Treatment Schedule

At the beginning of the chronic study, the high dose groups received the estimated maximum tolerated dose as determined in the 8-week subchronic study. The low dose was one-half of the high dose in all cases. In order to maintain the animals at the maximum doses that could be actually tolerated, body weight changes and survival were monitored, and, accordingly, doses were changed for the rats after 7 and 16 weeks of treatment, and for the mice after 12 weeks. To help assure survival until planned termination the dosing schedule was changed for rats to a cycle of 1 week of no treatment followed by 4 weeks of treatment. Seventy-eight weeks after the start of the test, dosing of rats and mice was stopped and observation of the animals continued until the test was terminated after 110 weeks for rats and 90 weeks for mice. This dosing schedule is outlined in Table III and Figures la and lb.

5.2 Observations

Individual body weights and food consumption per cage were recorded weekly for the first 10 weeks and monthly thereafter (Tables XVa, XVb, XVIa, and XVIb, and Figures 13a, 13b, 14a, and 14b, Appendix B) . Records of appearance, behavior, and signs of toxic effects were maintained at the same intervals as above. Animals were checked daily for mortality, and moribund animals were killed.

5.3 Necropsy

A gross necropsy was performed on each animal that died or was killed and on all survivors at termination. The weight of any discrete subcutaneous tissue mass was recorded. The dissection of rats and mice followed the same systematic technique whether the animals died or were killed. However, their blood smears for microscopic evaluation were prepared from the tail of each living animal immediately prior to injecting it with Diabutal^ intraperitoneally (0.3 to 0.5 ml for rats and 0.05 to 0.1 ml for mice). After induction of a state of anesthesia, the spinal cord and blood vessels at the back of the neck were severed with sharp-pointed scissors and the animal was exsanguinated and immediately necropsied.

The cervical lymph nodes, salivary glands, and thyroid with attached parathyroids, trachea, larynx, and esophagus (en bloc) were removed. The eyes, brain, pituitary, and nasal turbinates were removed, examined, and fixed. Thigh muscle and accompanying sciatic nerve and the femur were then excised, followed by abdominal skin (with mammary gland) and subcutaneous masses. The thoracic and abdominal cavities were then opened and the sternum was removed by cutting through the costochondral junctions. The thymus, heart (with small attached length of aorta), and lungs were removed. The lungs were fixed in their entirety. The thoracic spinal cord was removed. All lobes of the liver were taken including the free margin of each lobe. Any nodule or mass was represented in a block 10 x 5 x 3 mm cut from the liver and fixed in a marked capsule. The spleen was removed with a small piece of pancreas attached. The stomach was separated from the small intestines and esophagus and opened. Following removal of the stomach contents, its lining was examined. The mesenteric lymph nodes were

10 Table III. Dosage and Observation Schedule - Trichloroethylene Chronic Study Dose (mg Percent of Age at Treatment Time-Weighte d Dosage TCE/kg TCE in Dosinga Period Av. Dose" (mg Group Body Wt) Corn Oil (weeks) (weeks) TCE/k g Body Wt) Rats

Low dose 650 60.0 7 7C males and 750 60.0 14 9c females 500 60.0 23 14C 500 60.0 37 48d no treatment 85 32 549

High dose 1300 60.0 7 7C males and 1500 60.0 14 9c females 1000 60.0 23 14c 1000 60.0 37 48d no treatment 85 32 1097 Mice

Low dose 1000 15.0 5 6C males 1000 10.0 11 6C 1200 24.0 17 66C no treatment 83 12 1169

High dose 2000 15.0 5 6C males 2000 20.0 11 6C 2400 24.0 17 66C no treatment 83 12 2339

Low dose 700 10.0 5 12c females 900 18.0 17 66C no treatment 83 12 869

High dose 1400 10.0 5 6C females 1400 20.0 11 6C 1800 18.0 17 66C no treatment 83 12 1739

Matched controls received doses of corn oil by gavage calculated on the basis of the factor for the high dose animals (see section 5.1.3). aAge at initial dose or dose change. ^Time-weighted average dose = £ (dose in mg/kg x no. of days at that dose)/I (no. of days receiving any dose). In calculating the time-weighted average dose, only the days an animal received a dose are considered. cDosing 5 days per week each week. ^Dosing 5 days per week, cycle of 1 week of no treatment followed by 4 weeks of treatment. (Animals were treated for 38 weeks of the 48 week period.)

11 Figure la. Dosage Schedule - Chronic Study ­ Figure lb. Dosage Schedule - Chronic Study ­ Trichloroethylene-Treated Rats Trichloroethylene-Treated Mice excised along with a small amount of surrounding fatty tissue. The intestines were removed and straightened by cutting through their mesenteric attachment and were examined externally for abnormalities. Portions of duodenum, jejunum, ileura, and colon (about 1.5 cm long) were excised and placed in a capsule for fixation. If any gross lesion was noted from the serosal surface, the intestines were opened. After the selected portions were excised for separate preservation, the remaining portions of intestines and cecum were fixed in the container with the bulk of the tissues. The adrenals were removed with surrounding fatty tissue. After the kidneys were removed and their capsular surface freed of other tissues, they were bisected longitudinally and placed in tissue capsules. The urinary bladder was removed, a small amount of 10% formalin was injected if contracted, and it was opened slightly to examine the lining. The prostate and seminal vesicles were removed together. The testes with epididymides attached were fixed en bloc after the attached fat pad was removed. The ovaries, uterus, and vagina were removed and fixed en. bloc. The posterior 2 cm of rectum was removed with surrounding connective tissue and fixed en. mass.

Subcutaneous and other masses were removed and weighed. Location, size (dimensions or weight), color, consistency, and general appearance were recorded. Representative sections, 5 mm thick, were excised and fixed. If an animal had more than one mass or nodule, sections of each were placed in a marked capsule. If a mass involved the head, the entire skull was fixed after the cranial cavity had been opened.

The above protocol applied to animals necropsied after February 28, 1974, when the carcasses were no longer retained. Before that time, similar procedures were used, but fewer tissues were taken, and the carcasses were preserved.

5•4 Histological Preparation and Microscopic Examination

The necropsy was conducted by a dissector under the supervision of a pathologist. Each time a tissue was removed the presence of any gross lesion was recorded. All tissues were fixed in 10% buffered formalin (Appendix D, Tables XXVIIIa and XXVIIIb). The histologic technician trimmed the tissues under a fume hood to a thickness of 3-6 mm; any missing tissues or unusual lesions were brought to the attention of the pathologist. Tissues were dehydrated, cleared, and infiltrated on an Autotechnicon tissue processor, using a series of Technicon reagents (S-29 and UC-670) and Paraplast. Tissues were placed in a vacuum oven and subsequently embedded in Paraplast, using a Tissue Tek system. All blocks were sectioned at 5 microns; one slide was prepared from each block and stained with hematoxylin and eosin. Special staining procedures such as PAS, Trichrome, and PTAH were used occasionally as requested by the pathologist to diagnose a specific tumor cell type. All paraffin blocks were sealed with Paraplast and stored in plastic cabinets. Slides were boxed and delivered with all necropsy forms and work sheets to the pathologist at Hazleton Laboratories, Inc., for examination. Seven to 9 slides were prepared per mouse, and 7-10 per rat.

13 5.5 Data Processing and Confirmation

Summaries of the numbers of tissues examined for rats and mice are given in Tables XXVIIla and XXVIIlb. The pathologist recorded his findings into a dictaphone for the typist to transcribe on a computer data form, the Individual Animal Data Record (IADR). Information was included on the death of the animal, details of the necropsy, including physical abnormalities and tumors. IADRs were transmitted to EG&G/Mason Research Institute, Bethesda, MD, NCI operations contractor for the computerized system for collection, maintenance, and analysis of bioassay data. This system is known as the Carcinogenesis Bioassay Data System (CBDS) (Linhart et al. , 1974). Diagnoses of tumors and other animal abnormalities were coded using the coding system described in the "Systematized Nomenclature of Pathology" (SNOP), prepared by the Committee on Nomenclature and the Classification of Disease, College of American Pathologists, Chicago, 1965. The SNOP code has been modified for use in the Carcinogenesis Bioassay Data System. One code is entered for the topography or site of the lesion, and another for the diagnosis. Output from CBDS is in the form of the Individual Animal Pathology Report, which presents the complete pathology data for the animals within a group, including both tumor and non-tumor diagnoses. The initial output was reviewed and corrected by the pathologist at Hazleton. A further review of the findings was conducted by pathologists at Tracor Jitco, Inc., and the National Cancer Institute with special attention given to liver lesions. The differences in opinion were minor, and, in general, supported the diagnoses as presented. The final data are presented in the form as seen in Tables XXXIa, XXXIb, XXXIIa, and XXXIIb.

14 5.6 Positive Controls

In this bioassay carbon tetrachloride was administered as a positive control to both rats and mice, obtained from the same source and maintained under the same environmental conditions as the animals receiving trichloro­ ethylene. Solutions were prepared and administered by gavage in the manner described for trichloroethylene. Dosing was 5 times per week thoughout the study according to the dosage schedule in Table IV:

Table IV. Dosage and Observation Schedule - Carbon Tetrachloride Study Time-Weighted Dose Percent of Age at Treatment Av. Dose" (mg a Dosage (mg CCl^kg CCI4 in Dosing Period CCl4/kg Group Body Wt) Corn Oil (weeks) (weeks) Body Wt) Rats

Low dose males 25 2.5 6 10c 50 5.0 16 68C no treatment 84 32 47

High dose males 50 2.5 6 10c 100 5.0 16 68C no treatment 84 32 94

Low dose females 100 10.0 6 i4c 75 7.5 20 64C no treatment 84 32 80

High dose females 200 10.0 6 14c 150 7.5 20 64C no treatment 84 32 159 Mice

Low dose males 1250 25.0 5 78C and females no treatment 83 12 1250

High dose males 2500 25.0 5 78C and females no treatment 83 12 2500

Matched controls received doses of corn oil by gavage calculated on the basis of the factor for the high dose animals (see section 5.1.3). aAge at initial dose or dose change. ^Time-weighted average dose = £ (dose in mg/kg x no. of days at that dose)/Z (no. of days receiving any dose). In calculating the time-weighted average dose, only the days an animal received a dose are considered. cDosing 5 days per week each week.

15 6.0 CHRONIC TESTING: RESULTS - RATS

Sections 6.1 - 6.5 refer to trichloroethylene-treated rats and their vehicle-treated controls.

6.1 Body Weights

The range of the mean body weights for male rats was 193-194 g, and for female rats, 144-146 g, when placed on experiment (Tables XVa and XVb, Appendix B) . Weights in male rats peaked at 622 g at 46 weeks in control, 575 g at 46 weeks in low dose, and 535 g at 38 weeks in high dose animals. At these times there were, respectively, 20 control, 43 low dose, and 39 high dose animals. The average weights of surviving males in all groups were much lower than this at the termination of the test at 110 weeks. At this time the surviving 2 controls averaged 382 g, the 8 low dose males, 383 g, and the 3 high dose males, 423 g.

Average group weights of female rats peaked at 404 g at 62 weeks in control, 322 at 70 weeks in low dose, and 326 g at 94 weeks in high dose animals. At these times there were, respectively, 17 control, 23 low dose, and 20 high dose animals. The average weights of female rats were also lower at termination at 110 weeks. At this time, 8 surviving controls averaged 326 g, and 13 low dose and 13 high dose females both averaged 311 g. These decreases in average weights may reflect weight losses in individual animals and/or a mortality pattern where larger animals died sooner.

6 .2 Clinical Observations

During the first year of the study, the appearance and behavior of the treated rats were generally comparable with the controls except that occasionally hunched appearance and discoloration of the fur of the lower abdomen by urine stains were noted in a few test animals as early as week 2. Respiratory involvement characterized by labored breathing, wheezing, and/or reddish nasal discharge was noted in both treated and control groups, and increased as the animals aged.

Adverse clinical signs in all treatment groups were noted at a low or moderate incidence during the first year, and with gradually increasing frequency in the treated animals during the second year of the study. These signs included hunched appearance; roughening of the haircoat; eyes squinted or showing a reddish discharge; localized alopecia on extremities or body; sores, particularly on the tail; and stains on the haircoat.

6.3 Survival

Data for rats are given in the individual pathology tables in Appendix D. The methodology for statistical analysis is described in Appendix C. Data for statistical analysis are summarized in Tables XVIIIa and XVIIIb, Appendix C. Tables of results are also in Appendix C.

A high proportion of rats died during the experiment. For males, 17/20 control, 42/50 low dose, and 47/50 high dose animals died prior to

16 scheduled termination. For females, 12/20 control, 35/48 low dose, and 37/50 high dose animals died before scheduled termination. (Two low dose females were missing and were not counted in the denominator for that group.)

Survival probabilities were estimated by the product-limit procedure of Kaplan and Meier (1958) (Tables XXa and XXb and Figures 2a and 2b). The estimated probabilities (standard errors) of survival to 110 weeks were 0.100 (0.067) for male control, 0.140 (0.049) for male low dose, 0.060 (0.034) for male high dose, 0.400 (0.110) for female control, 0.252 (0.061) for female low dose, and 0.260 (0.062) for female high dose.

The survival times of vehicle control, low dose, and high dose groups of rats were compared (Table XXc). Among male rats, the age-adjusted test for linear trend (Tarone, 19 75) is significant at P = 0.001, and the high dose vs. control test and the high dose vs. low dose test are significant at P = 0.001, indicating that high dose male rats died earlier than low dose and control male rats and that earlier death is associated with higher dose. Among female rats, the low dose group died earlier than the control group, as shown by a test with P = 0.028. The high dose female rats died earlier than control female rats (P = 0.049), but slightly, and not significantly, later than the low dose female rats. The dose-response test, therefore, shows only P = 0.117.

6.4 Pathology

A variety of neoplastic and non-neoplastic lesions were recorded among control, low dose, and high dose rats. Tumors in specific organ systems by site of origin and by anatomic site are summarized in Tables XXIXa (page 121) and XXIXb (page 124), and pathologic observations for individual animals are listed in Tables XXXIa (page 135) and XXXIb (page 147) in Appendix D.

There are no significant differences In t_he incidences o_f total j^ujnorjs or of a. specific ^umoj: type .between treated and control rats. As seen in Table V, there is no indication of a treatment-related effect. While the percentage of tumor-bearing animals is actually lower in treated animals, this is likely related to the decrease in their survival.

Table V. Tumor Incidence - Rats with Tumors

Males Females Low High Low High Control Dose Dose Control Dose Dose

Before 110 weeks 4/17 5/42 4/47 4/12 6/35 4/37 At 110 weeks 1/3 2/8 1/3 3/8 6/13 8/13 Total 5/20 7/50 5/50 7/20 2/48 12/50 Percent 20% 14%- 10% 35% 25% 24%

17 TIME ON STUDY (WEEKS)

Figure 2a. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Male Rats 18 Figure 2b. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Female Rats 19 Incidences of the most common tumor types according to those dying before and at 110 weeks are presented in Table XXII (page 102) in Appendix C. The tumors observed are described in the following paragraphs.

Primary malignant renal tumors were observed in 4 rats, 2 of which were unilateral malignant mixed tumors of the kidney in male controls (010, 019). One of these (010) also had a hamartoma in the outer medulla of the opposite kidney. A similar hamartoma affected the outer renal medulla of one of the low dose males (002) . The hamartomas contained an admixture of adipose tissue, spindle cells, and tubular structures with ciliated columnar epithelium. The malignant mixed tumors consisted primarily of proliferating fat cells and spindle cells mushrooming from the renal medulla through the cortex to form a large extrarenal protuberance. Mitoses were abundant and frequently abnormal. The proliferating tissues were invasive and contained as an integral component small tubular nests of proliferating epithelial tissue. Thus, the 2 renal hamartomas and the 2 malignant mixed tumors contained the same cellular elements. They appeared to arise in or near the renal medulla. The only other primary renal tumor was an adenocarcinoma arising from nephronic epithelium in a low dose male (015).

The relationship between the renal hamartomas (2 rats) and renal malignant mixed tumors (one of the same rats and another) remains to be clarified. The hamartomas clearly arose near the corticomedullary junction, the mixed tumors apparently so. The similarities of site of origin and tissue components suggest that they were developmentally related. The hamartoma was probably congenital and may have undergone malignant transformation to the malignant mixed tumor. The mixed tumor had structural similarities comparable to nephroblastoma, a well known tumor in many species including human beings, swine, and others. Because the mixed tumors in these older rats had both epithelial and nonepithelial components and were obviously highly aggressive, the term malignant mixed tumor is considered suitable.

Primary tumors of the thyroid were found in 5 animals. Of these, 2 were benign follicular-cell adenomas found in a control male and a low dose male. Three malignant follicular-cell adenocarcinomas were also found in a low dose male, a high dose male, and a high dose female.

No pituitary or mammary tumors were seen in the male rats. Among the females, however, chromophobe adenomas and a benign tumor of the pituitary were found in 4/20 controls, 2/47 low dose, and 6/49 high dose groups. Mammary fibroadenomas were found in 3/20 controls, 5/45 low dose, and 7/48 high dose female rats. Multiple mammary fibroadenomas were found in one low dose female (050), and 3 high dose females (023, 030, and 033). The only other primary mammary tumor was an adenocarcinoma in a control female (014) . Three other tumors of the reproductive system were found, an ovarian granulosa-cell carcinoma in a control female (009) and 2 sarcomas of the endometrium in a low dose female (026) and a high dose female (037) . The sarcomas were rather poorly differentiated and a more specific classification is not considered feasible.

There were several other miscellaneous malignant neoplastic entities of epithelial origin, each occurring in a different animal. One control male

20 (007) had a primary pulmonary carcinoma with both glandular and squamous differentiation; multiple metastases with similar biphasic differentiation were present in the lungs, cervical lymph node, and kidneys. This was a very aggressive tumor. A low dose male (Oil) had a squamous cell carcinoma in the axillary region. A high dose male (034) had a pilomatrixoma of the skin and another (014) had a massive aortic body tumor at the base of the heart. One low dose female (023) had an adrenal cortical carcinoma.

Two rats had fibromas of the subcutis, a low dose male (025) and a low dose female (013). Malignant tumors included a fibrosarcoma of the subcutis in a low dose male (004) and a malignant anaplastic giant-cell tumor in the abdomen of a control male (002) . The latter is the only tumor of this type in any rat on the study. A low dose female (032) had a subcutaneous liposarcoma. Hemangiosarcomas were recognized in a control male, a low dose male, 2 high dose males, and a low dose female.

Tumors of hematopoietic type were limited to reticulum-cell sarcoma, a malignant tumor which affected 1 control female (004), 1 low dose female (016), and 1 high dose female (006).

No non-neoplastic lesions appeared to be related to treatment with the exception of renal changes. Slight to moderate degenerative and regenerative tubular alterations, primarily affecting proximal tubular epithelium, were common in treated rats but lacking in controls. However, chronic renal disease occurred frequently among aged treated and control rats. A high incidence of chronic respiratory disease was observed among the rats without any apparent difference in type, severity, or morbidity as to sex or group. No significant toxic hepatic changes were observed.

6.5 Tumor Probabilities

For the purpose of statistical analysis, letters, or marks, were assigned to sets of pathologic diagnoses (Table XVII). Frequencies of the more commonly observed marks are summarized in Table XXII, Appendix C.

The probabilities of observing histopathologic diagnoses among control, low dose, and high dose groups of rats were estimated. See Appendix C for a description of methods of estimation and statistical testing. Tests were performed for reticulum-cell sarcoma, lymphosarcoma, or malignant lymphoma (mark b), fibroadenoma of the mammary gland (mark g), hemangioma of any site (mark h), follicular adenocarcinoma of the thyroid (mark p), and chromophobe adenoma of the pituitary (mark t). Since neither hepato­ cellular carcinoma of the liver (mark a) nor adenoma and carcinoma of the lung (mark c and d) was observed in any rat, as they were in mice, tests comparing these marks were not performed. Test results are shown in Tables XXIIIa-e. None of the tests demonstrates increases in the probability of observing a^ tumor for dosed rats over control rats.

21 6.6 Controls

6.6.1 Survival

Table VI shows a comparison of the survival of the rats receiving carbon tetrachloride and trichloroethylene with pooled colony controls at 78 and 110 weeks:

Table VI. Comparison of Survival of Colony Controls and Trichloroethylene- and Carbon Tetrachloride-Treated Rats

Trichloroethylene Carbon Tetrachloride Controls Low Dose High Dose Low Dose High Dose Interval M F M F M F M F M F

Initial 100 100 50 50 50 50 50 50 50 50 78 weeks 67 75 31 20 12 23 34 38 34 21 110 weeks 26 51 8 13 3 13 14 26 7 14

At both time periods a slightly greater number of rats survived which had received carbon tetrachloride than those which had received trichloro­ ethylene. Survival among the controls was generally greater.

6.6.2 Tumors

The incidence of both hepatocellular carcinoma and neoplastic nodule in colony controls and in rats receiving carbon tetrachloride is given in Table VII:

Table VII. Incidence of Liver Tumors ­ Colony Control and Carbon Tetrachloride-Treated Rats Hepatocellular Neoplastic Animal Group Carcinoma Nodule

Males controls 1/99 0/99 low dose 2/50 2/50 high dose 2/50 1/50

Females controls 0/98 2/98 low dose 4/49 2/49 high dose 1/49 3/49

A low incidence of both hepatocellular carcinoma and neoplastic nodule was found in both colony controls and carbon tetrachloride-treated rats. Neither of these lesions was found in any of the rats receiving trichloro­ ethylene. Statistical tests show no difference between control and trichloroethylene-treated animals (Tables XXIVa and XXIVb). However, the comparison of observed hepatocellular carcinomas in carbon tetrachloride- treated low dose female rats compared with pooled vehicle-treated female controls is significant•by a one-tailed Fisher exact test (P = 0.011) (see Table XXIVc). When observed hepatocellular carcinomas and neoplastic nodules are analyzed together by the same test, there are significantly

22 more lesions among both the carbon tetrachloride-treated male rats (P = 0.033) and female rats (P = 0.009) than among respective male and female pooled controls (see Table XXIVd). Individual animal data for carbon tetrachloride-treated rats are given in Tables XXXIIIa-d, Appendix D.

In the carbon tetrachloride-treated rats marked hepatotoxicity with resultant fibrosis, bile duct proliferation, and regeneration was observed. The majority of liver nodules observed were diagnosed as being regenerative rather than neoplastic. These regenerative nodules were composed of hepatocytes which were generally larger and paler staining than the adjacent hepatic parenchyma. These nodules were multiple and circumscribed by mature fibrous connective tissues.

The lesions diagnosed as neoplastic nodules contained hepatocytes which varied in appearance from large pale-staining cells to smaller, more basophilic cells with a disorganized pattern, poorly defined sinusoids, and essential absence of portal triads. The hepatocytes composing the neo­ plastic nodules were much more variable in appearance than those of the regenerative nodules.

The diagnosis of hepatocellular carcinoma was based on the presence of less organized architecture and more variability in the cells comprising the neoplasms. Often the neoplastic cells were arranged in thickened cell plates or occasionally in an acinar pattern. Several of the carcinomas had a prominent vascular supply as opposed to the neoplastic and regenerative nodules.

23 7.0 CHRONIC TESTING: RESULTS - MICE

Sections 7.1 - 7.6 refer to trichloroethylene-treated mice and their vehicle-treated controls.

7.1 Body Weights

Weights of male mice averaged 17 g, and female mice, 14 g, when placed on experiment (Tables XVIa and XVIb). Average weights in male mice peaked at 34 g at 34 weeks in all groups and average weights in female mice peaked at 27 g at 34 weeks. Survivors of both sexes maintained approximately these respective weights until termination of the experiment after 90 weeks.

7.2 Clinical Observations

During the first year of the study, the appearance and behavior of the treated and control mice were generally comparable. Alopecia (generalized and/or localized), sores on the tail and other parts of the body, and a hunched appearance were noted in an increasing number of mice, mostly males, in all groups beginning on week 14 and persisting during the study.

After 50 weeks of treatment, bloating or abdominal distention was the predominant observation in the high dose males. By week 74, approximately 50% of all treated males had a bloated appearance which persisted until they died or were killed after 90 weeks. A few treated females also showed abdominal distention prior to termination. Subsequent necropsy of the animals confirmed the presence of liver tumors.

7.3 Survival

Data for mice are given in the pathology tables in Appendix D. Data for statistical analysis are summarized in Tables XlXa and XlXb, Appendix C. Tables of results are also in Appendix C.

Some mice died before the end of the experiment at 90 weeks from other than accidental causes. For males, 12/20 controls, 14/50 low dose, and 28/50 high dose died before termination. For females, 0/20 controls, 8/50 low dose, and 8/47 high dose died. (Three high dose females were missing and were not counted in the denominator for that group.)

Survival probabilities were estimated by the product-limit procedure of Kaplan and Meier (1958) (Tables XXIa and XXIb, Figures 3a and 3b). The estimated probabilities (standard errors) of survival of mice to the end of the chronic test at 90 weeks were 0.400 (0.110) for male control, 0.715 (0.064) for male low dose, 0.409 (0.070) for male high dose, 1.000 (0.000) for female control, 0.835 (0.053) for female low dose, and 0.830 (0.055) for female high dose.

The survival of control, low dose, and high dose groups of mice was compared (Table XXIc). The age-adjusted test for linear trend (Tarone, 1975) among male mice is marginally significant (P = 0.096), but high dose is not significantly different from control. High dose male mice lived

24 40 50 60 70 TIME ON STUDY (WEEKS) Figure 3a. Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Male Mice 25 30 40 50 60 70 TIME ON STUDY (WEEKS) Figure 3b, Product-Limit Estimates of Probability of Survival - Trichloroethylene-Treated Female Mice 26 significantly shorter lives than low dose male mice (P = 0.001), but control male mice also had significantly shorter lives than low dose male mice (P = 0.004). Among female mice, low dose and high dose groups are not significantly different, but mice in both groups had significantly shorter lives than the control group (P = 0.035). The age-adjusted test for linear trend yields P = 0.068, only marginally significant.

7.4 Pathology

A variety of neoplastic and non-neoplastic lesions were observed among control and treated mice. Tumors in specific organ systems by site of origin and by anatomic site are summarized in Tables XXXa (page 127) and XXXb (page 130), and individual animal pathology is listed in Tables XXXIIa (page 158) and XXXIIb (page 168) in Appendix D.

As presented and discussed in section 7.5, highly significant differences in the incidences of primary malignant tumors of the liver, i.e., hepato­ cellular carcinomas, were found between treated and control grjou_ps. Hepato­ cellular carcinoma was observed in 1/20 control males, 26/50 low dose males, 31/48 high dose males, 0/20 control females, 4/50 low dose females, and 11/47 high dose females (Table XXV). Metastasis of the hepatocellular carcinoma to the lung, looked for on the basis of single sections., occurred in 4/50 low dose males, and in 3/48 high dose males. One control male (019) with hepatocellular carcinoma died during week 72 of the study. Among the low dose males the first hepatocellular carcinoma was observed in a mouse (029) that died during week 81 and the first metastasis was in one (007) that died during week 88. Among the high dose males the first hepato­ cellular carcinoma was observed in a mouse (046) that died during week 27; 10 mice that died on or before week 78 had hepatocellular carcinoma and the first metastasis was in a mouse (035) that died during week 83. Hepato­ cellular carcinoma was found only in females killed at termination at 90 weeks. Thus, the incidence

The hepatocellular carcinoma varied in size and number among the affected mice. The diagnosis was based on size of neoplasm, histologic appearance, and the presence of metastasis. The tumors varied from those composed of well differentiated hepatocytes in a relatively uniform trabecular arrange­ ment to rather anaplastic lesions in which mitotic figures occurred in cells which varied greatly in size and tinctorial characteristics. Many of the tumors were characterized by the formation of relatively discrete areas of highly anaplastic cells within the tumor proper which were, in turn, surrounded by relatively well differentiated neoplastic cells. In general, various arrangements of hepatocellular carcinoma occurred, as described in the literature, including those with an orderly cord-like arrangement of neoplastic cells, those with a pseudoglandular pattern resembling adeno­ carcinoma, and those composed of sheets of highly anaplastic cells with minimal cord or gland-like arrangement. Multiple metastatic lesions were observed in the lung, including several neoplasms which were differentiated and relatively benign in appearance. The morphology of these tumors is illustrated in Figures 4-10.

27 Figure 4. Primary hepatocellular carcinoma, mouse (high dose male #32). The rather well differentiated tumor of trabecular pattern has a recognizable boundary (->) with the pre­ existent hepatic tissue. Rematoxylin and eosin, x75 Figure 5. Same as Figure 4, x300

28 29 Figure 6. Primary hepatocellular carcinoma, mouse (high dose male #26). The rather anaplastic solid tumor has a recognizable boundary (->) with the pre-existent hepatic parenchyma. Mitoses are abundant. Hematoxylin and eosin, x240

Figure 7. Primary hepatocellular carcinoma, mouse (high dose male #15) with both solid trabecular pattern and papillary pattern. Most cells are large and resemble normal hepatocytes. There are also smaller cells with less cytoplasm and smaller, more basophilic nuclei. These cells usually occur in clusters and are especially prominent in the larger papillary structures.

30 31 Figure 8. Primary hepatocellular carcinoma, mouse (high dose male #32). There is an area of highly anaplastic cells in a papillary pattern contiguous with an area of well differentiated cells in a trabecular pattern. Hematoxylin and eosin, x120 Figure 9. Secondary hepatocellular carcinoma, lung, mouse (high dose male #15). The metastatic hepatocellular carcinoma has invaded and extended into a bronchiole. The bulk of the tumor consists of rather well differentiated hepatocytes but there are scattered foci of smaller, more anaplastic, basophilic, neoplastic cells (->) also. Hematoxylin and eosin, x120 Figure 10. Metastatic hepatocellular carcinoma, lung, mouse (high dose male #15). Well differentiated hepatocytes comprise a large nodule (edge at upper left) and invade perivascularly at lower center. Numerous foci of smaller, more anaplastic, basophilic, neoplastic cells occur in vessels (->) and alveolar capillaries, and invade perivascularly. Hematoxylin and eosin, x96

32 33 In addition to hepatocellular carcinomas, malignant lymphoid tumors and pulmonary tumors appeared somewhat elevated although not significantly at the 0.05 level. Malignant lymphoid tumors (reticulum-cell sarcoma and lymphosarcoma) were recognized in 1/20 control males, 4/47 low dose males, 2/45 high dose males, 1/19 control females, 5/49 low dose females, and 6/47 high dose females. The number of tissues involved varied among the different cases.

Benign fibrous tumors consisted of fibroma of the subcutis in a low dose male (002) and neurofibroma in skeletal muscle of the back of a high dose male (044) . Malignant tumors included fibrosarcoma of the skin or subcutis in 3 of 20 control males. A high dose male (034) that died after 90 weeks on study had a primary fibrosarcoma within the abdominal cavity with multiple metastases. Among the females, one low dose mouse (003) killed terminally had uterine fibrosarcoma. A control female (008) had a highly vascular osteosarcoma of the soft tissues of the back and a high dose male (043) had a hemangiosarcoma of the lung.

Of the respiratory tumors, benign pulmonary adenomas were diagnosed in 5/50 low dose males, 1/48 high dose males, 3/50 low dose females, and 5/47 high dose females. Alveolar adenocarcinoma, a malignant tumor, was diagnosed in 1/48 high dose males, 2/49 low dose females, and 2/47 high dose females. The one in the high dose male (035) had metastasized to regional lymph nodes, periaortic tissues, and the skin of the chest.

Other benign tumors included adenoma of the Harderian gland in 1 low dose male, 1 low dose female, and 2 high dose females. An adenoma of tubular origin was found in the kidney of a high dose male (013), and an ovarian cystadenoma in a low dose female (043) . Other carcinomas included an endometrial adenocarcinoma in a control female (003), an ovarian granulosa­ cell carcinoma in a low dose female (033), and a mammary adenocarcinoma in a low dose female (038).

7.5 Tumor Probabilities

See Appendix C for a description of the statistical tests and tables of the results.

Frequencies of the more commonly observed marks are summarized in Table XXV. Estimated probabilities of observing histopathologic diagnosis among control, low dose, and high dose groups of mice were compared. Tests were conducted for hepatocellular carcinoma (mark a), reticulum-cell sarcoma, lymphosarcoma, or malignant lymphoma (mark b), carcinoma or adenocarcinoma of the lung or alveoli (mark c), adenoma of the lung (mark d), and carcinoma, adenocarcinoma, or adenoma of the lung (mark c or d) . Test results are shown in Tables XXVIa-e. No mouse was observed to have fibroadenoma of the mammary gland (mark g) or chromophobe adenoma of the pituitary (mark t), so tests comparing these marks were not performed as they had been for the rats.

34 The age-adjusted tests for linear trend (Tarone, 1975) were highly significant for hepatocellular carcinoma in male mice (P < 0.001) and female mice (P = 0.002). The tests of high dose vs. control are also highly significant in both male mice (P < 0.001) and female mice (P = 0.008). A similar test of low dose vs. control shows significant differences for male mice (P = 0.004) and female mice (P = 0.090). Esti­ mated probabilities of observing hepatocellular carcinoma in male mice, depending on the week on study in which they died, are displayed in Table IX and are graphed in Figure 11. In male mice, the tumors were observed earlier in the high dose group than in the low dose group, which may result from earlier mortality among high dose male mice. At the end of the study at 90 weeks, the estimated probability (standard error) of observing hepatocellular carcinoma in male mice was 0.938 (0.043) for high dose, 0.683 (0.076) for low dose, and 0.077 (0.074) for control. In female mice, no tumors were observed before terminal sacrifice at 90 weeks; therefore, no graphic presentation is included. At 90 weeks the estimated probabilities (standard errors) of observing hepatocellular carcinoma in female mice were 0.282 (0.072) for the high dose, 0.100 (0.047) for the low dose, and 0.000 (0.000) for the controls. Figure 12 shows a comparison of the percentage of animals of either sex with observed hepatocellular carcinoma. These data are summarized in Table VIII:

Table VIII. Incidence of Hepatocellular Carcinoma - Trichloroethylene-Treated Mice

Males Females

Controls 1/20 0/20 Low dose 26/50 (P = 0.004) 4/50 (P = 0.090) High dose 31/48 (P < 0.001) 11/47 (P = 0.008)

Test for significant difference of each group from controls. In addition, age-adjusted tests for linear trend were also highly significant for male mice (P < 0.001) and female mice (P = 0.002).

None of the tests of other marks showed significance at levels of 0.05 or less. However, the age-adjusted tests for linear trend for carcinoma or adenocarcinoma of the lung (mark c) may indicate a relationship with treat­ ment at a level P = 0.109 for male mice and P = 0.225 for female mice. Sim­ ilarly, the comparison of mark b, sarcoma, lymphosarcoma, and lymphoma, between high dose and control female mice may indicate a relationship (P = 0.172).

7.6 Colony Controls

The incidence of hepatocellular carcinomas for other control groups main­ tained in the same room as trichloroethylene is given in Table X. Groups A-D, which include the trichloroethylene-matched control group, were treated with corn oil. The incidence of hepatocellular carcinomas in the trichloroethylene-matched control group was typical of that observed in

35 Table IX. Estimated Probabilities of Observing Hepatocellular Carcinoma - Trichloroethylene-Treated Male Mice

Control Low Dose High Dose j n n' P j n n' P j n n' P 0 20 20 .000 0 50 50 .000 0 48 48 .000 32 20 20 .000 16 50 50 .000 13 48 48 .000 39 19 19 .000 18 49 49 .000 14 47 47 .000 60 18 18 .000 31 48 48 .000 15 46 46 .000 61 17 17 .000 51 47 47 .000 24 44 44 .000 64 16 16 .000 53 46 46 .000 25 42 42 .000 66 15 15 .000 58 45 45 .000 27 40 39 .025 68 14 14 .000 63 44 44 .000 28 39 38 .050 72 13 12 .077 65 43 43 .000 29 37 37 .050 76 12 12 .077 77 41 41 .000 30 36 35 .076 77 10 10 .077 81 40 39 .025 36 34 33 .104 78 9 9 .077 86 38 37 .051 42 33 33 .104 90 8 8 .077 88 37 36 .076 53 32 31 .132 90 35 12 .683 60 31 31 .132 j = Week on study 61 29 28 .162 70 28 27 .191 n = No, of animals 71 27 26 .221 alive at beginning 74 26 25 .251 of the week 75 25 25 .251 78 24 23 .283 n' = No. of animals 83 23 22 .314 without observed 88 22 21 .345 tumor at the end 90 21 2 .938 of the week P = Kaplan-Meier estimate of the probability of observed tumor No comparable figure is presented for female mice because no tumors were observed before terminal sacrifice at 90 weeks.

TIME ON STUDY (WEEKS) Figure 11. Product-Limit Estimates of Probability of Observing Hepatocellular Carcinoma - Trichloroethylene-Treated Male Mice 37 Figure 12. Comparison of Incidence of Hepatocellular Carcinoma in Trichloroethylene-Treated Male and Female Mice other control groups, thus the significance of heptocellular carcinoma in trichloroethylene-treated mice remained similar when compared with the pooled colony controls.

Table X. Incidence of Hepatocellular Carcinoma - Colony Control Mice

Control Median Males Females Group Birth Date Vehicle Untreated Vehicle Untreated

Aa 7-17-72 1/20 0/20 B 3-20-72 1/18 0/20 C 5/31/72 1/19 1/20 D 10/12/72 2/20 0/20 E 3/27/72 1/19 0/19 F 6/15/72 2/18 0/19 G 10/27/72 2/18 2/20 H 10/04/72 0/15 0/18

Total 5/77 5/70 1/80 2/76

aMatched controls for trichloroethylene.

7.7 Positive Controls

Few mice receiving carbon tetrachloride survived until the planned termina­ tion of the test, compared with a considerable number in each of the trichloroethylene-treated groups as shown in Table XI:

Table XI. Comparison of Survival in Colony Controls - Vehicle-Treated and Trichloroethylene- and Carbon Tetrachloride-Treated Mice

Trichloroethylene Carbon Tetrachloride Controls Low Dose High Dose Low Dose High Dose Interval M F M F M F M F M F

Initial 77 80 50 50 48 47 50 50 50 50 78 weeks 53 71 40 41 23 21 11 10 2 4 90 weeks 38 65 35 40 40 39 0 0 0 1

Hepatocellular carcinomas were found in practically all mice receiving carbon tetrachloride, including those dying before termination of the test. The incidence of liver tumors was considerably greater in carbon tetra­ chloride-treated mice than in trichloroethylene-treated mice as shown in Table Xlla. Individual animal data for carbon tetrachloride-treated mice are given in Tables XXXIVa-d, Appendix D.

39 Table Xlla. Comparison of Hepatocellular Carcinoma Incidence in Colony Controls - Vehicle-Treated and Trichloroethylene- and Carbon Tetrachloride-Treated Mice

Trichloroethylene Carbon Tetrachloride Animals Controls Low Dose High Dose Low Dose High Dose

Males 5/11 26/50 31/48 49/49 47/48

Females 1/80 4/50 11/47 40/40 43/45

These liver tumors in carbon tetrachloride-treated mice varied greatly in appearance from lesions which contained well differentiated hepatic cells that had a relatively uniform arrangement of the cords to very anaplastic liver cells having large hyperchromatic nuclei, often with inclusion bodies, and with vacuolated, pale cytoplasm. Arrangement of the neoplastic liver cells varied from short stubby cords to nests of hepatic cells and occasionally acinar arrangements. Mitotic figures were often present. Some of the tumors were characterized by discrete areas of highly anaplastic cells surrounded by relatively well differentiated tumor cells. The neoplasms occurring in the treated mice were similar in appearance to those noted in the trichloroethylene-treated mice.

The test week at which the first animal died in which a hepatocellular carcinoma was observed in each group is given in Table Xllb:

Table Xllb. Comparison of Time (weeks) to Liver Tumor Detection in Colony Controls - Vehicle-Treated and Trichloroethylene­ and Carbon Tetrachloride-Treated Mice

Trichloroethylene Carbon Tetrachloride Animals Controls Low Dose High Dose Low Dose High Dose

Males 72 81 27 48 26

Females 90 90 91 16 19

In addition to the higher incidence, hepatocellular carcinomas were observed much earlier in carbon tetrachloride-treated mice than in the trichloroethylene-treated mice. Tumors in control mice were observed much later.

40 8.0 DISCUSSION

Trichloroethylene is one among a series of halogenated chemicals tested in the National Cancer Institute carcinogenesis bioassay program. The results clearly indicate that exposure to trichloroethylene has resulted in hepatocarcinogenicity in both sexes of the B6C3F1 mouse. No evidence of such activity was apparent in the rat tests.

8•1 Design of Bioassay

8.1.1 Selection of Animal Species and Strain

Since it is well known that species vary in their carcinogenesis response to chemicals, two species are routinely used in the carcinogenesis bioassay program to decrease the possibility of false negative results based on tests in a resistant strain of animals. A clearly positive result in only one test species is a valid indication of the carcinogenic activity of the test compound while failure to detect carcinogenicity in another species only indicates a lower susceptibility of the latter under the conditions of the test.

Rats and mice were selected as the test species because they are genetic­ ally standardized, readily available species that reproduce well and can be easily, successfully, and economically maintained. They have been the most extensively used species for carcinogenesis testing.

The Osborne-Mendel strain of rats was chosen because of the experience gained by the Food and Drug Administration where the strain was used for many years as a general purpose test animal. In addition, it had been found by Reuber and Glover (1970) to be sensitive to the carcinogenicity of carbon tetrachloride by subcutaneous administration. The B6C3F1 strain of mice, an Fl hybrid cross of the C57BL/6 female with the C3H/He male, was chosen because it had been extensively and satisfactorily used in the NCI carcinogenesis bioassays.

8.1.2 Route of Exposure

Trichloroethylene was administered by gavage in this study while the main human exposures are by inhalation of vapors and by ingestion through contaminated water and food products. The selected route of exposure is considered relevant to all modes of human exposure because trichloro­ ethylene is readily absorbed and distributed to all organs following ingestion or inhalation. This has been shown in several species, including the rabbit (Gasq, 1936), the dog (Barrett and Johnston, 1939), and the guinea pig (Fabre and Truhaut, 1952). The fact that in this study tumor induction occurred in the liver indicates that trichloroethylene was absorbed and that systemic exposure of tissues occurred.

41 8.1.3 Selection of Doses and Duration of Exposure

An attempt was made to select the highest dose that could be administered for most of the animal's lifetime without altering the animal's normal longevity from effects other than carcinogenicity (estimated maximum tolerated dose). A lower dose, corresponding to one half of the high dose, was also tested to assure that at least one group would survive well through the test period if unpredicted toxic effects occurred in the high dose group.

The selection of doses for bioassay is made to assure the greatest probability of detecting a carcinogenic effect within an experimental protocol which must use relatively small numbers of animals compared with the large number of human beings exposed.

8.1.4 Methodology

The protocol and methodology of this bioassay generally conformed to recognized methodology for assessing carcinogenicity as adopted by the NCI. While the NCI has revised in several respects its test procedures since the time the present tests were designed (Guidelines for Carcinogenesis Bioassay in Small Rodents, 1975), these changes do not reduce the meaning­ fulness of this study. The major changes in current NCI protocols as compared with the test procedures used in this study are: (a) More complete and longer prechronic studies are carried out in an effort to predict the MTD more accurately. Prechronic studies are now routinely conducted for 90 days. Histopathological studies are performed on animals from all dose groups, necessary to establish a maximum tolerated dose. Clinicopatho­ logical tests are also performed when indicated. Experience has shown that more extensive prechronic tests reduce or eliminate the need to change dose levels during the course of the chronic study. In the past, survival and slight decrease in weight gain were used as the main indicators of the MTD. (b) Larger matched control groups are now also included with each test so that pooling of controls from several compounds is not necessary. In this bioassay 20 matched controls for each sex and species were started. How­ ever, 18 chemicals in the case of the mice and 5 chemicals in the case of rats were tested concurrently, and pooling of the controls for the various chemicals, in order to have a larger number of colony control animals, was anticipated.

It has been recommended by several panels on carcinogenesis testing (Berenblum, 1969; FDA Advisory Committee, 1971; Golberg, 1974) that administration of a food additive in a carcinogenesis test might begin prior to conception and continue in the offspring because such treatment would provide a more thorough examination of the carcinogenic potential of test compounds. The present bioassay makes no attempt to measure either transplacental or neonatal carcinogenic effects.

Commercial laboratory diets were fed to the test animals. Recommendations have been made to use semi-synthetic diets of known and constant composi­ tion as a step toward uniformity in interpretation of experimental results from different laboratories and to decrease contamination with mycotoxins, pesticides, or other agents. However, problems in preparation and storage,

42 nutritional adequacy, palatability, and handling have precluded so far their adoption for practical use in these large scale lifetime studies.

8.2 Test Compound Purity

The purity of the trichloroethylene used in the chronic bioassay was greater than 99%, as determined by gas chromatographic total area data and infrared spectra (see Appendix A). In the gas chromatographic analysis, the percentage of each component was determined from the relative area of its gas chromatographic peak. The sum of the area comprising the peaks was considered to total 100%. In the final analytical work, in which the minor components were actually identified, standard samples of each component were used to correct for differences in gas chromatographic detector response. Nonvolatile materials, such as polymers and inorganic salts, would not have been detected by gas chromatography but there is no reason to suspect their presence. Any significant quantity of such impurities would have been detected in the infrared spectra, which, in fact, compare well with reference trichloroethylene spectra.

Inhibitors and stabilizers are commonly added to trichloroethylene used for vapor degreasing. Some of the trace components, identified in Batch #4 by a combination of gas chromatography and mass spectrometry in subsequent analyses, are 1,2-epoxybutane (0.19%), ethyl acetate (0.04%), epichloro­ hydrin (0.09%), N-methylpyrrole (0.02%), and diisobutylene (0.03%). According to a major manufacturer, these are typical inhibitors in commercial formulations of trichloroethylene used for vapor degreasing.

It is reported that trichloroethylene in the mid-1960s contained substantial quantities of 1,1,2,2-tetrachloroethane, a highly toxic precursor in the acetylene-based manufacturing process, but current technology and the switch to ethylene-based processes now result in a highly pure commercial product (NIOSH, 1973). That conclusion is consistent with the analytical data on the batches used for the bioassay. Analytical work on bioassay Batch #4 confirmed the absence of detectable quantities of 1,1,2,2,-tetrachloroethane (<5 ppm) and 1,1,1,2-tetra­ chloroethane (<2 ppm).

While the results obtained in the present bioassay could possibly have been influenced by an impurity in the trichloroethylene used, the extremely low amounts of impurities found make this improbable.

8.3 Metabolism, Distribution, and Excretion

The metabolism, distribution, and excretion of trichloroethylene in humans, rats, and mice was reviewed in Criteria for a Recommended Standard... Occupational Exposure to Trichloroethylene (1973), issued by the National Institute for Occupational Safety and Health.

The data describing evidence for the metabolic pathways of trichloro­ ethylene in mice are not as extensive as that available for man and rats. However, the data do indicate similar metabolic pathways for this compound in the 3 species and do not support the concept that metabolic differences among these species might explain the lack of carcinogenic response of the

43 rat to trichloroethylene as compared with the mouse. Recent data on trichloroethylene metabolism in man are consistent with earlier findings in experimental animals (Ertle et a1., 1972; Ikeda et al., 1971, 1972; Ikeda and Ohtsuji, 1972; Ikeda and Inamura, 1973; Kimmerle and Eben, 1973; Nomiyama and Nomiyama, 1971; Parkhouse, 1969; Soucek and Vlachova, I960; Stewart and Dodd, 1964; Vignoli et al., 1970).

8.4 Toxicology

Depression of the central nervous system is the primary acute toxic effect of trichloroethylene in both humans and animals. This effect was demonstrated in rats by Adams et al. (1951) in short term toxicity studies. In humans, nausea and vomiting, headache, vertigo, dizziness, tinnitis, unsteady walk, fatigue, sleepiness, and even excitement have all been reported in patients and in those persons inadvertently or occupationally exposed. This and other acute and subchronic toxicity of the compound are extensively reviewed in Criteria for a Recommended Standard...Occupational Exposure to Trichloroethylene (1973) issued by the National Institute for Occupational Safety and Health.

Although data on chronic or carcinogenesis studies were not available in the published literature prior to this report, data from subchronic studies indicated that the liver and kidney were target sites of the toxic effects of trichloroethylene.

The effects of this chemical on liver and kidney were noted by Adams et al. (1951) in subchronic inhalation studies using rats as an experimental model. Liver and kidney weights of both male and female rats were increased but no histopathological abnormalities were reported. Increased liver weights were also observed in both guinea pigs and rabbits. In mice, tri­ chloroethylene is not as nephrotoxic as several other chlorinated methane, ethane, and ethylene derivatives (Plaa and Larson, 1965). However, the hepatotoxicity of trichloroethylene has been reported by several authors. It is much less severe than that of carbon tetrachloride, an intensively studied chlorinated methane that was used as a positive control in this bioassay. In a review of trichloroethylene von Oettingen (1955) described hepatic damage which was reported as early as 1932 in dogs. He also quotes Fiessinger and Laur in 1936 as observing "centrolobar degeneration and vacuolization, and in some animals, a picture similar to yellow atrophy of the liver". Clinical dysfunction of the liver in dogs was reported by Seifter (1944). Mice have shown hepatic dysfunction when exposed to trichloroethylene by inhalation for up to 8 weeks (Kylin et al. , 1965) or by intraperitoneal injection (Klaassen and Plaa, 1966). In other studies, the hepatotoxicity of trichloroethylene was enhanced by pretreatment with acetone or isopropyl alcohol (Traiger and Plaa, 1974).

Thus, hepatic damage of varying degrees has been found following repeated exposures of short duration to trichloroethylene. These observations establish the need for chronic studies.

44 8.5 Pathology and Survival

8.5.1 Rats

In the bioassay various neoplastic entities, both benign and malignant, occurred in all rat dosage groups and controls. Some are rather rare types of tumors in rats, such as pilomatrixoma of skin and aortic body tumor, but none was significantly related to treatment with trichloroethylene. No toxic hepatic change nor primary hepatic tumor was observed in the rats.

Chronic respiratory disease occurred without regard to sex or treatment group in rats. The only treatment-related lesion was a chronic nephropathy encountered among most rats of both sexes and at both high and low doses of the compound. This nephropathy was characterized by degenerative and regenerative changes in tubular epithelium with questionable interstitial response. The lesion is dissimilar from the chronic nephropathy so commonly encountered in rats with advancing age and recognized in some rats on this experiment. No treatment-related lesions severe enough to appear responsible for death were detected in the rats. Nevertheless, decreased survival was generally dose-related.

Only a low incidence of both neoplastic nodule and hepatocellular carcinoma was observed in rats receiving carbon tetrachloride, the positive control compound. Survival was slightly better among the carbon tetrachloride- treated than among the trichloroethylene-treated rats.

In this bioassay as in other previous carcinogenic bioassays of chlorinated organic compounds, some chemicals have been identified as liver carcinogens in the mouse, but have produced no observed carcinogenic effect in the rat. The difference in susceptibility to the induction of tumors by certain chemicals in the rat as compared to the mouse may be attributed to many factors and is beyond the scope of these studies.

8.5.2 Mice

In the mice, there was a significant increase in the incidence of hepato­ cellular carcinomas in both low and high dose males and high dose females (P < 0.05) and low dose females (P = 0.09). The tumors were found in 12 of 27 mice dying during the experiment and in 19 of 21 high dose male mice killed at termination of the experiment. Low dose male mice as well as high and low dose female mice had good survival rates. With the exception of 3 low dose male mice dying at 81 to 88 weeks, hepatocellular carcinoma was found only in those animals killed at the end of the experiment. The lower probability of tumor observation in female mice may reflect either the lower doses they received or an actual sex difference in response. Thus, trichloroethylene was found to have a dose-related carcinogenic effect on the liver of both sexes of mice. No other tumor was related to treatment.

Only 20 matched vehicle control mice were started for each species and sex in this bioassay; however, the significance of hepatocellular carcinoma in trichloroethylene-treated mice remains similar when compared with data from control mice for other halogenated solvents tested concurrently.

45 In this study the doses of trichloroethylene administered to the test animals resulted in a greater survival of mice than of rats. The increased mortality in rats may in part have resulted from intercurrent disease as well as from a greater sensitivity to the toxic effects of trichloro­ ethylene.

In the positive control group, hepatocellular carcinoma was observed in practically all mice receiving carbon tetrachloride. Only one of these animals survived until planned termination of the test. Except for high dose male mice, hepatocellular carcinomas were observed at an earlier age among carbon tetrachloride-treated than among trichloroethylene-treated mice. Thus, carbon tetrachloride was a much stronger hepatocarcinogen than was trichloroethylene, under the conditions of these tests.

8.6 Effect of Various Compounds in the Same Room

Several halogenated solvents were being tested simultaneously in the same room, i. e., 5 in the room housing rats and 18 in the room housing mice. This is not expected to change the results significantly; however, no experimental studies of cross contamination or simultaneous administration are available. A protective effect from simultaneous exposure to other, and halogenated solvents would not be expected, and it is highly unlikely that an interaction of possible airborne contaminant amounts of solvents would bring about false positives, considering the high doses of trichloro­ ethylene used.

The species in which tumors were found, i.e., mice, were housed in a room where 17 other chemicals were being tested; however, stringent precautions against cross contamination were employed. The mice were kept in cages with filter tops which limit the amount of expired chemical in the air available for inhalation by other animals, the total air in each room was changed 10 to 15 times per hour, and the mouse racks were transported to another room with a large hood for the daily intubations. Furthermore, the hepatocarcinomas in mice were present at a greater than P = 0.01 level of significance and were produced by doses of trichloroethylene of 700 to 2400 mg/kg, which are several thousand-fold greater than any possible con­ tamination could have been. A dose-related effect was observed and, any possible chemical in the general room air did not affect controls. Thus, although this room arrangement is not desirable as is stated in the Guidelines for Carcinogen Bioassay in Small Rodents, there is no evidence the results would have been different with a single compound in a room.

8.7 Relationship to the Toxicity of Carbon Tetrachloride

Carbon tetrachloride was used as a positive control because of its demonstrated ability to produce liver tumors in rats, hamsters, and mice (Reuber and Glover, 1970, Delia Porta et al., 1961, Eschenbrenner and Miller, 1946). The doses used were approximately 10-fold less than for trichloroethylene in rats, but were only slightly higher for males and 50% higher for females than for trichloroethylene in mice.

Pathology of the liver was evident in carbon tetrachloride-treated rats. Hepatocellular carcinomas and neoplastic nodules were found in a few rats

46 of both dose groups and sexes in contrast to the results with trichloroethylene where no tumors and very little non-tumor pathology of the liver were reported. This is particularly significant since the dose of trichloroethylene used was approximately 10 times greater than for carbon tetrachloride.

Hepatocellular carcinomas were found in practically all mice including those dying before termination of the test. The incidence was considerably greater than for trichloroethylene-treated mice (see section 7.7).

Although in mice the MTD values of carbon tetrachloride and trichloro­ ethylene were similar, all except one carbon tetrachloride-treated animal (of both sex and dose groups) died prior to termination at 90 weeks. The survival of the trichloroethylene-treated female mice was excellent, and even 40% of the high dose males survived until termination of the test. Death in the carbon tetrachloride-treated animals could have resulted either from toxicity or carcinogenicity since tumors were observed in practically all animals. These results confirm previous work. Both Klaassen and Plaa (1966) and Gehring (1968) have shown that in mice the hepatotoxicity of carbon tetrachloride is much greater than that of trichloroethylene, both on an absolute basis and in relation to anesthetic effects and to the LDso value.

8.8 Conclusions

The administration of trichloroethylene under the experimental conditions described in this report induced a high incidence of hepatocellular carcinoma in B6C3Fl mice of both sexes. The test in rats is inconclusive: large numbers of rats died prior to planned termination; in addition, the response of this rat strain to the hepatocarcinogenicity of the positive control compound, carbon tetrachloride, appeared relatively low. Although direct extrapolation to man is not possible, the identification, using this methodology, of trichloroethylene as a carcinogen in animals serves as a warning of its possible carcinogenicity in humans.

47 9.0 BIBLIOGRAPHY

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Armitage, P. (1971) Statistical Methods in Medical Research, Chapter 14, John Wiley and Sons, New York, NY.

Barrett, H. M. ; Johnston, J. H. (1939) The Fate of Trichloroethylene in the Organism, J^ Biol^ ChenK_ 127:765-770.

Berenblum, I. (Ed.) (1969) Carcinogenicity Testing; A Report of the Panel on Carcinogenicity of the Cancer Research Commission of the UICC, UICC Technical Report Series, Vol. 2, International Union Against Cancer, Geneva.

Chemical Economics Handbook (1972) Chemical Information Services, Stanford Research Institute, Menlo Park, CA.

Christensen, H. E. ; Luginbyhl, T. T. (Eds.) (1974) The Toxic Substances List, 1974 Ed., USDHEW, National Institute for Occupational Safety and Health, Rockville, MD.

Cochran, W. G. (1954) Some Methods for Strengthening the Common Chi-square Tests, Biometrics jLO:417-454.

Cox, D. R. (1972) Regression Models and Life-Tables (with discussion), J^ Roy. Statist. Soc. B34:184-220.

Deichmann, W. B.; Gerarde, H. W. (1969) Toxicology of Drugs and Chemicals, Academic Press, New York, NY.

Delia Porta, G.; Terracini, B; Shubik, P. (1961) Induction with Carbon Tetrachloride of Liver Cell Carcinomas in Hamsters, JN_ Natl. Cancer Inst. 2^6:855-863.

Dowty, B. ; Carlisle, D.; Laseter, J. L.; Storer, J. (1975) Halogenated Hydrocarbons in New Orleans Drinking Water and Plasma, Science 187:75-77.

Dreisbach, R. H. (1974) Handbook of Poisoning: Diagnosis and Treatment, 8th Ed., Lange Medical Publications, Los Altos, CA.

Ertle, T.; Henschler, D.; Mueller, G.; Spassowski, M. (1972) Metabolism of Trichloroethylene in Man - I. The Significance of Trichloroethanol in Long-term Exposure Conditions, Arch. Toxicol. 29:171-188.

48 Eschenbrenner, A. B.; Miller, E. (1944) Studies on Hepatomas. I. Size and Spacing of Multiple Doses in the Induction of Carbon Tetrachloride Hepatomas, J^ Natl^ Cancer Inst. 4:385-388.

Eschenbrenner, A. B.; Miller, E. (1946) Liver Necrosis and the Induction of Carbon Tetrachloride Hepatomas in Strain A Mice, J^ Natl. Cancer Inst, 6:325-341.

Fabre, R. ; Truhaut, R. (1952) Contribution a I/etude de la Toxicologie du Trichlorethylene - II. Resultats des Etudes Experimentales chez L'animal, Brit. J^ Ind._ Med. 9^:39-43.

Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis Report on Cancer Testing in the Safety Evaluation of Food Additives and Pesticides (1971) Toxicol. Appl. Pharma^ol^ 2^:419-438.

Food and Drug Administration (1974) Code of Federal Regulations, Title 21, 121.1041.

Frear, D. E. H. (Ed.) (1969) Pesticide Index, 4th Ed., College_Science Publishers, State College, PA, p. 377.

Gasq, M. (1936) Etudes Toxicologie et Hygienic des Solvents Chlores Acethyleniques, Delmar, Bordeaux.

Gehring, P. J. (1968) Hepatotoxic Potency of Various Chlorinated Hydro­ carbon Vapors Relative to their Narcotic and Lethal Potencies in Mice, Toxicol. Appl. Pharmacol. U:287-298.

Golberg, L. (1974) Carcinogenesis Testing of Chemicals, Proceedings, Conference on Carcinogenesis Testing in the Development of New Drugs, May 23-25, 1973, Washington, DC, CRC Press, Inc., Cleveland, OH.

Ikeda, M. ; Imamura, T. (1973) Biological Half-life of Trichloroethylene and Tetrachloroethylene in Human Subjects, Int. AL^LL Arbetsmed. 31--2Q9-224.

Ikeda, M.; Ohtsuji, H. (1972) A Comparative Study of the Excretion of Fujiwara Reaction-Positive Substances in Urine of Humans and Rodents Given Trichloro- and Tetrachloro-Deri'vatives of Ethane and Ethylene, Brit. J^ Ind. Med. 29:99-104.

Ikeda, M.; Ohtsuji, H.; Imamura, T.; Komoike, Y. (1972) Urinary Excretion of Total Trichloro-Compounds, Trichloroethanol and Trichloroacetic Acid as a Measure of Exposure to Trichloroethylene and Tetrachloro­ ethylene, Bjrit^ J^_ Ind. Med. 29:328-333.

49 Ikeda, M.; Ohtsuji, H.; Kawai, H.; Kuniyoshi, M. (1971) Excretion Kinetics of Urinary Metabolites in a Patient Addicted to Trichloroethylene, Brit. J^ Ind. Med^ 28:203-206.

International Labour Office (1972) Encyclopedia of Occupational Health and Safety, Vol. II, McGraw-Hill, New York, NY.

Kaplan, E. L.; Meier, P. (1958) Nonparametric Estimation from Incomplete Observations, J^ Amer^ Statist. Ass. 53:457-481.

Kimmerle, G.; Eben, A. (1973) Metabolism, Excretion and Toxicology of Tri­ chloroethylene after Inhalation, Arch. Toxicol. 30:127-138.

Klaassen, C. D.; Plaa, G. L. (1969) Comparison of the Biochemical Alterations Elicited in Livers from Rats Treated with Carbon Tetrachloride, Chloroform, 1,1,2-Trichloroethane, and 1,1,1-Trichloro­ ethane, Biochem. Pharmacol. 18:2019-2027.

Klaassen, C. D.; Plaa, G. L. (1966) Relative Effect of Various Chlorinated Hydrocarbons on Liver and Kidney Function in Mice, Toxicol. Appl. Pharmacol. 9^:139-151.

Kylin, B. ; Sumegi, I.; Yllner, S. (1965) Hepatotoxicity of Inhaled Tri­ chloroethylene and Tetrachloroethylene, Long-term Exposure, Acta Pharmacol. Toxicol. 2^2:379-385.

Linhart, M. S.; Cooper, J.; Martin, R. L.; Page, N.; Peters, J. (1974) Carcinogenesis Bioassay Data System, Computers Biomed. Research 7_:230-248.

Mantel, N. (1963) Chi-square Tests with One Degree of Freedom: Extensions of the Mantel-Haenzel Procedure, J^ Amei^ Statist. Ass. 58:690-700.

Mantel, N. (1966) Evaluation of Survival Data and Two New Rank Order Statistics Arising in Its Consideration, Cancer Chemother. Rep. 5(0:163-170.

National Cancer Institute (March 1975) Guidelines for Carcinogen Bioassay in Small Rodents, TR-1, Division of Cancer Cause and Prevention, National Institutes of Health, Bethesda, MD.

National Institute for Occupational Safety and Health (1973) Occupational Exposure to Trichloroethylene, USDHEW, Public Health Service, Washington, DC.

Nomiyama, K.; Nomiyama, H. (1971) Metabolism of Trichoroethylene in Humans. Sex Difference in Urinary Excretion of Trichloroacetic Acid and Trichloroethanol, Int. Arch. Arbeitsmed. 28:37-48.

50 Parkhouse, J. (1969) Uptake and Metabolism of Trichloroethylene. Can. Anaesthetists* Soc. J^ 16:113-118.

Patty, F. A. (Ed.) (1963) Industrial Hygiene and Toxicology, Vol. II, D. W. Fassett and D. D. Irish (Eds.), Interscience Publishers, New York, NY.

Plaa, G. L.; Larson, R. E. (1965) Relative Nephrotoxic Properties of Chlorinated Methane, Ethane, and Ethylene Derivatives in Mice, Toxicol. Appl. Pharmacol. 2:37-44.

Price, H. L.; Dripps, R. D. (1965) General Anesthetics, II. Volatile Anesthetics: Diethyl Ether, Divinyl Ether, Chloroform, Halothane, Methoxyflurane, and other Halogenated Volatile Anesthetics, The Phar­ macological Basis of Therapeutics, 3rd Ed., L. S. Goodman and A. Gilman (Eds.), MacMillan, New York, NY, pp. 83-99.

Rechnagel, R. 0.; Glende, E. A.; Jr. (1973) Carbon Tetrachloride Hepatotoxicity: An Example of Lethal Cleavage, Crit. Rev. Toxicol. 2:263-297.

Reuber, M. D.; Glover, E. L. (1970) Cirrhosis and Carcinoma of the Liver in Male Rats Given Subcutaneous Carbon Tetrachloride, J^_ Natl. Cancer Inst. 44:419-423.

Saffiotti, U.; Montesano, R.; Sellakumar, A. R.; Cefis, F.; Kaufman, D. G. (1972) Respiratory Tract Carcinogenesis in Hamsters Induced by Different Numbers of Administrations of Benzo(a)pyrene and Ferric Oxide, Cancer Res. 32:1073-1081.

Sax, N, I. (1968) Dangerous Properties of Industrial Materials, 3rd Ed., Van Nostrand Reinhold, New York, NY, p. 1180.

Seifter, J. (1944) Liver Injury in Dogs Exposed to Trichloroethylene, J^ Ind. Hyg. Toxicol. 2^6:250-252.

Soucek, B.; Vlachova, D. (I960) Excretion of Trichloroethylene Metabolites in Human Urine, Brit. J^_ Ind. Med. 17:60-64.

Stecher, P. G. (Ed.) (1968) The Merck Index, 8th Ed., Merck & Co., Inc., Rahway, NJ, p. 1069.

Stewart, iL D.; Dodd, H. C. (1964) Absorption of Carbon Tetrachloride, Trichloroethylene, Tetrachloroethylene, Methylene Chloride, and 1,1,1-Trichloroethane through the Human Skin, Amer. Ind. Hyg. Ass. J. 25:439-446.

Tarone, R. E. (1975) Test for Trend in Life-Table Analysis, Biometrika, 62:679-682.

51 Traiger, G. J.; Plaa, G. L. (1974) Chlorinated Hydrocarbon Toxicity: Potentiation by Isopropyl Alcohol and Acetone, Arch. Environ. Health 2^:276-278.

Vignoli, L.; Jouglard, J.; Vignoli, P.; Terrasson de Fougeres, P. (1970) Acute Intoxication by Trichloroethylene Ingestion. Metabolism of this Poison, Med_^ Leg. Assicuorazioni 1_8:789-798. von Oettingen, W. F. (1955) The Halogenated Hydrocarbons: Toxicity and Potential Dangers, PHS Publication No. 414, U. S. Government Printing Office, Washington, DC, pp. 203-227.

Wiseman, P. (1972) Introduction to Industrial Organic Chemistry, Applied Science Publishers Ltd., Essex, England.

52 APPENDIX A: CHEMISTRY

53 Chemical and Physical Characteristics

Physical state: Liquid, nonflammable (Patty, 1963; Sax, 1968) Odor: Resembling chloroform (Patty, 1963) Melting point: -73°C (Patty, 1963) Boiling point: 86.7°C (760 mm Hg) (Stecher, 1968) Solubility: 0.1 g/100 ml water at 20°C; soluble in ether, ethyl alcohol, and chloroform (Patty, 1963); dissolves most fixed and volatile oils (Stecher, 1968) Odor threshold: 24.1 ppm Specific gravity: 1.45560 (25°/4°C) (Patty, 1963) Vapor density: 4.54 (air = 1) (Patty, 1963) Vapor pressure: 77 mm Hg (25°C) (International Labour Office, 1972) Refractive index: 1.4777 (20°C) (Patty, 1963) Percent in "saturated" air: 10.2 (25°C) (Patty, 1963) Handling precautions: Use with adequate ventilation (Stecher, 1968). Must be stored in sealed, light-resistant containers.

Technical Product and Impurities

Tetrachloroethane has been reported as an impurity in technical trichloro­ ethylene, particularly in that produced by the acetylene-based process (Dreisbach, 1974; NIOSH Criteria Document, 1973). The chemical used for this bioassay contained no detectable quantity of tetrachloroethane.

Manufacturing Processes

In one industrial process, trichloroethylene is produced from acetylene. The process involves addition of chlorine to acetylene to give 1,1,2,2­ tetrachloroethane. The more common process since 1972 involves the addition of chlorine to ethylene to give ethylene dichloride and then further chlorination to 1,1,1,2-tetrachloroethane, followed by HC1 elimination to yield trichloroethylene (Wiseman, 1972).

54 Chemical Analysis

Trichloroethylene Batch #1 - January 9, 1973*

Gas Chromatography (Table Al)

Detector: Flame ionization Recorder range: 1 mv full scale Column: 3' x 1/4" od, aluminum, 80-100 mesh Porapak Q Temperatures (°C): Injection port 205, detector 250, column oven pro­ grammed from 60 (2 min) to 205 (20 min) at 6°/min Flow rates (ml/min): Nitrogen carrier 45, hydrogen 45, air 475 Attenuation: From 1 x 16 to 103 x 32 Remarks: About 0.8 yl sample was injected *Conducted by Hazleton Laboratories, Inc.

Table Al. Analysis of Total Area Data - Batch #1

Component By Retention Time Area Total Areaa Percent (min) (cm2) (cm2) (A/At x 100) Av. (%)

2.8 0.1 A 0.00067 3.4 0.1 B 0.00082 0.001 3.3 0.1 C 0.00075

13.3 0.54 A 0.0036 13.6 0.34 B 0.0031 0.003 13.8 0.41 C 0.0034

18.0 118 A 0.79 18.0 72 B 0.65 0.7 18.4 83 C 0.69

19.2 8 A 0.053 19.4 6 B 0.054 0.06 19.8 7 C 0.058

21. lb 14,840 A 99.1 20.9b 10,920 B 99.2 99.1 21.3b 11,900 C 99.1

26.0 13 A 0.087 26.0 9 B 0.082 0.09 25.5 12 C 0.10

Total 100.0

a A = 14,980 B = 11,010 C = 12,000 ^Trichloroethylene

Infrared Spectroscopy (Figure Al) 55 14 l-ijl J jtl>?|tliUi t-M|^ M I Mllljiiljifj

Figure Al. Infrared Spectrum of Trichloroethylene Trichloroethylene Batch #3 - February 15, 1974*

Gas Chromatography (Table A2)

Same as for Batch #1, except as follows:

Flow rates (ml/min): Hydrogen 60, air 450 Attenuation: From 1 x 16 to 10^ x 128 Remarks: About 1.0 yl sample was injected ^Conducted by Hazleton Laboratories, Inc.

Table A2. Analysis of Total Area Data - Batch #3

Component By Retention Time Area Total Areaa Percent (min) (cm2) (cm2) (A/At x 100) Av. (%)

12.7 3. 6 A 0.01 12.6 3.3 B 0.01 0.01 12.7 3. 9 C 0.01

16.8 305 A 0.63 16.8 284 B 0.62 0.6 17.0 343 C 0.68

18.2 55 A 0.11 18.1 52 B 0.12 0.1 18.2 63 C 0.13

20.2b 48,200 A 99.0 20.2b 45,400 B 99.0 99.0 20.2b 49,800 C 99.0

24.2 23 A 0.05 24.2 21 B 0.05 0.05 24.2 25 C 0.05

26.0 86 A 0.18 26.0 79 B 0.17 0.2 26.1 81 C 0.16

Total 100.0

a A = 48,670 B = 45,840 C = 50,320 ^Trichloroethylene

Infrared Spectroscopy (Figure A2)

57 00

Figure A2* Infrared Spectrum of Trichloroethylene Trichloroethylene Batch #4 - July 8, 1974*

Gas Chromatography (Table A3)

Same as for Batch #1, except as follows:

Temperatures (°C): Injection port 215, detector 240 Flow rates (ml/min): Hydrogen 65, air 450 Attenuation: From 1 x 16 to 10^ x 16 Remarks: About 1.2 yL sample was injected ^Conducted by Hazleton Laboratories, Inc.

Table A3. Analysis of Total Area Data - Batch #4

Component By Retention Time Area Total Areaa Percent (min) (cm2) (cm2) (A/At x 100) Av. (%)

20.7 1.7 A 0.001 20.7 1.3 B 0.001 0.001 20.6 1.7 C 0.001

22.8 440 A 0.38 22.7 370 B 0.36 0.4 22.7 410 C 0.37

24.1 280 A 0.24 23.9 230 B 0.22 0.2 23.9 250 C 0.22

26.4b 115 ,500 A 99.4 26.2b 104 ,300 B 99.4 99.4 26.2b 111 ,300 C 99.4

34.8 37 A 0.03 34.8 33 B 0.03 0.03 34.7 34 C 0.03

Total 100.0

a A = 116,250 B = 104,930 C = 112,000 ^Trichloroethylene

Infrared Spectroscopy (Figure A3)

59 Figure A3, Infrared Spectrum of Trichloroethylene Identification of Trace Components

July 10, 1975 Bioassay Batch #4*

Analysis Techniques

Temperature-programmed vapor-phase chromatography (vpc) was performed on trichloroethylene in order to obtain relative areas and retention times of the major component and impurities present in the compound. Vapor-phase chromatography-mass spectrometry was performed to obtain the mass peaks of the components present. Vapor-phase chromatography was then repeated on the sample spiked with compounds which had the correct mass and fragmen­ tation properties to confirm enhancement of the impurity peaks. In addi­ tion, vpc with electron capture detection was used to analyze for trace amounts of the tetrachloroethanes.

Results - Vapor-Phase Chromatography

Survey System 1: Experimental conditions were as follows: Bendix 2500, 1.8 x 4 mm id Chromosorb column, flame ionization detection, oven tempera­ ture programmed from 100 to 250°C at 10°/min.

A major peak and 5 impurities were detected.

Retention Retention Time Area (Relative Possible Peak Time (min) (Relative to TCE Peak) to TCE Peak) Identity

1 7.0 0.70 0.003 unknown 2 7.9 0.79 0.4 1,2-epoxybutane 3 8.6 0.85 0.3 ethyl acetate 4 10.1 1.00 100 trichloro­ ethylene 5 11.5 1.14 0.05 N-methylpyrrole 6 12.9 1.28 0.06 diisobutylene

*Conducted by Midwest Research Institute, Kansas City, MO.

Survey System 2: Experimental conditions: Tracor MT 220, 5% Carbowax 20M TRA on 60-80 mesh Gas-Chrom Q, 1.8 m x 4 mm column, flame ionization detec­ tion, oven temperature 40°C.

A major peak and 2 impurites were detected.

Retention Retention Time Area (Relative Possible Peak Time (min) (Relative to TCE Peak) to TCE Peak) Identity

4 2.4 1.00 100.00 trichloro­ ethylene 5 6.0 2.50 0.27 N-methylpyrrole 7 10.9 4.54 0.80 epichlorohydrin

61 Quantitation and Identity System 1 (Epoxybutane): Experimental conditions: Tracor MT 220, Chromosorb 102, 1.8 m x 4 mm id column, flame ionization detection, oven temperature 100°C.

By addition of known standards to the trichloroethylene sample, it was determined that peak 2 was not due to ethyl vinyl ether, ethyl acetate, tetrahydrofuran, methyl ethyl ketone, or 1,2-epoxyisobutane. However, addition of an authentic sample of 1,2-epoxybutane enhanced the peak. The epoxybutane in trichloroethylene was quantitated against a known standard of epoxybutane.

Retention Quantitation Peak Time (min) Identity (%)

1 - unknown 2 18.11 1,2-epoxybutane 0.19 3 22.52 ethyl acetate

Quantitation and Identity System 2 (Ethyl Acetate): Experimental condi­ tions: Bendix 2500, Chromosorb 102, 1.8 m x 4 mm id, flame ionization detection, oven temperature 135°C.

The vpc-mass data indicated that peak 3 could be ethyl acetate. Addition of an authentic sample of ethyl acetate to the trichloroethylene enhanced this peak. Ethyl acetate in the trichloroethylene was quantitated using an ethyl acetate standard.

Retention Quantitation Peak Time (min) Identity (%)

1 5.7 unknown 2 7.8 1,2-epoxybutane 3 9.8 ethyl acetate 0.04

Quantitation and Identity System 3 (N-Methylpyrrole and Trimethylpentene): Experimental conditions: Tracor MT 220, Chromosorb 102, 1.8 m x 4 mm id, flame ionization detection, oven temperature 175°C.

The vpc-mass data indicated that peak 5 could be N-methylpyrrole. Addition of an authentic sample of ltf-methylpyrrole to the trichloroethylene enhanced this peak.

The vpc-mass data indicated that peak 6 could be diisobutylene (2,2,4-tri­ methylpentene) . Addition of an authentic sample of 2,2,4-trimethylpentene enhanced this peak.

Peaks 5 and 6 were quantitative against the authentic standards.

62 Retention Quantitation Peak Time (min) Identity (%)

4 4.0 trichloroethylene 5 6.5 N-methylpyrrole 0.02 6 10.0 trimethylpentene 0.03

Quantitation and Identity System 4 (Epichlorohydrin); Experimental condi­ tions: Tracor MT 220, 5% Carbowax 20M TRA on 60-80 mesh Gas-Chrom Q, 1.8 m χ 4 mm, flame ionization detection, oven temperature 40°C.

The vpc-mass data indicated that peak 7 could be epichlorohydrin. Addition of an authentic sample of epichlorohydrin enhanced this peak. The epi­ chlorohydrin present in the trichloroethylene sample was quantitated using an epichlorohydrin standard. On the Chromosorb 102 column, epichlorohydrin has the same retention time as trichloroethylene.

Retention Quantitation Peak Time (min) Identity (%)

4 trichloroethylene 5 8.19 N-methylpyrrole 7 11.18 epichlorohydrin 0.09

Quantitation and Identity System 5 (Tetrachloroethane): Experimental con­ ditions: Tracor MT 220, Chromosorb 102, 1.8 m χ 4 mm id, 63Ni electron capture detection, oven temperature 190°C.

On injection of a 6 μΐ neat sample of trichloroethyene, Lot No. 063014, no peaks were detected at the retention times of either tetrachloroethane isomer. The concentration of tetrachloroethane in the trichloroethylene (Lot No. 063014) is therefore less than 2 ppm for the 1-, 1,1,2-isomer and less than 5 ppm for the 1,1,2,2-isomer.

Note: A fresh sample of trichloroethylene (Aldrich Lot No. 090947) did contain a peak at the same retention time as 1,1,1,2-tetrachloroethane. Quantitation against known 1,1,1,2-tetrachloroethane indicated a concen­ tration of 3 ppm.

Minimum Retention Detectable Standard Time (min) Amount (ng)

1,1,1,2-Tetrachloroethane 12.0 0.1 1,1,2,2-Tetrachloroethane 16.7 0.3

63 Vapor-Phase Chromatography-Mass Spectrometry: Experimental conditions: Varian MAT CH-4B interfaced via a Watson-Biemann helium separator to a Micro-Tek 2000 MF gas chromatograph, data processed by Varian 620/i com­ puter, Chromosorb 102 column, 1.8 m x 4 mm id. See Table A4.

Conclusions

Six small impurities were detected in trichloroethylene (Lot No. 063014) by vapor-phase chromatography. Mass spectrometry data indicated that the 5 larger impurities were 1,2-epoxybutane, ethyl acetate, Nhmethylpyrrole, diisobutylene, and epichlorohydrin. Trichloroethylene samples spiked with authentic samples of the impurities showed enhancement of the corresponding peaks. Quantitation studies indicated the following impurity concen­ trations: 1,2-epoxybutane, 0.19%; ethyl acetate, 0.04%; _N-methylpyrrole, 0.02%; diisobutylene, 0.03%; epichlorohydrin, 0.09%. The first impurity peak (0.003%, relative area) was not detected by mass spectrometry. 1,1,2,2- and 1,1,1,2-tetrachloroethane were not detected by electron capture detection or mass spectrometry; the levels of minimum detection on the electron capture detector for the 2 tetrachloroethane isomers were 5 and 2 ppm, respectively.

64 Table A4. Va2or-Phase Chromato8ra2h~-Mass SJ2ectrometr~

Literature Values for: 1,2-EJ20X~butaneb Intensity Relative Intensity Relative fea!&a Mass to Base Peak Mass to Base Peak

2 28 (N2) 100 42 92 42 100 41 92 41 93 27 58 27 39 72 28 72 34 29 32 29 30 39 35 39 28 71 36 71 28 57 20 57 20 Eth~l Acetateb 3 43 100 43 100 29 43 29 25 27 23 27 13 45 49 45 13 61 49 61 10 42 16 42 6 70 30 70 5 26 8 26 4 88 15 b N-Meth~l2~rrole 5 81 93 81 100 80 59 80 80 39 24 39 37 42 18 42 30 53 27 53 28 28 100 (N2) 28 17 55 13 55 17 27 52 27 15 b Diisobut~lene

6 57 100 57 100 55 88 55 42 97 47 97 29 29 51 29 22 56 51 56 19 112 31 112 15 27 17 27 11 69 24 69 9 EJ2ichloroh~drinb

7 57 100 57 100 27 100 27 39 29 79 29 31 49 88 49 25 31 65 31 22 62 52 62 18 28 100 (Nz) 28 16 51 25 51 8

aPeak notation the same as previously indicated. hEight Peak Index of Mass SJ2ectra, Vol. I (1970), Mass Spectrometry Data Centre, AWRE, Aldermaston, Reading, RG7 4PR, United Kingdom.

65 Manufacturer's Feed Analysis (Wayne Lab-Blox Meal) (Allied Mills Inc., Chicago, IL 60606)

Crude protein (Min.) 24 .0%

Crude fat (Min.) 4.0%

Crude fiber (Max.) 4.5%

Ingredients

Animal liver meal Fish meal Dried whey Corn and wheat flakes Ground yellow corn Soybean meal Wheat middlings Cane molasses Soybean oil Brewer's dried yeast Vitamin A palmitate Irradiated dried yeast (source of vitamin D) D-Activated animal sterol Vitamin E supplement Menadione sodium bisulfite (source of vitamin K activity) Riboflavin supplement Niacin Calcium pantothenate Choline chloride Thiamine Ground limestone Dicalcium phosphate Salt Manganous oxide Copper oxide Iron carbonate Ethylene diamine dihydriodide Cobalt carbonate Zinc oxide

66 Basal Feed Analysis

A study of apparent pesticide residues in basal feed samples was conducted at Gulf South Research Institute under contract to NCI on November 10, 19 72. Three samples of feed used at Hazleton were analyzed. No lindane, heptachlor, aldrin, heptachlor epoxide, endrin, DDD, chlordane, methoxy­ chlor, toxaphene, or organophosphate was found. Two of the samples contained 0.00392 and 0.00682 ppm DDE, and 2 contained 0.00629 and 0.02016 ppm DDT. One sample contained 0.03955 ppm Aroclor 1254. The 3 samples contained 0.00249, 0.00141, and 0.00167 ppm dieldrin. The method of analysis was as follows:

A 20 g sample was extracted with 150 ml 6% diethyl ether in hexane on a 25 mm od x 40 cm chromatographic column containing 1" Na2S(>4. With the stopcock closed, 25 ml extracting solvent was added. The feed sample was added and allowed to settle. The column was filled with extracting solvent and the sample again was allowed to settle. The stopcock was opened and 150 ml extracting solvent was collected in a 500 ml standard taper round- bottom flask. A Synder column was placed on the flask and the extracting solvent was evaporated on a 65-70°C heating mantle. The Snyder column was rinsed with 30 ml hexane and the extract was reduced to about 10 ml. Evaporation continued and the Snyder rinse was repeated a second time. When the extract again was reduced to 10 ml, the heat was removed and the extract was reduced to about 2 ml under vacuum.

The extracted sample was cleaned up on a 25 mm od x 40 cm chromatographic column containing 5" Florisil (activated 15 hours at 135°C) and topped with 1/2" Na2SO4. The sample extract was quantitatively transferred to the column with a small portion of hexane. The following extracting solvents were passed through the column and collected:

Fraction A: 175 ml hexane

Fraction B: 200 ml 6% diethyl ether in hexane

Fraction C: 225 ml 15% diethyl ether in hexane

Fraction D: 200 ml 30% diethyl ether in hexane

Each fraction was evaporated as before almost to dryness and the residue was quantitatively transferred to a 10 ml volumetric flask and diluted to volume using hexane. An aliquot was taken for gas-liquid chromatographic analysis. Compounds were identified according to chromatographic retention times only and, therefore, should be considered tentative.

67 Water Analysis (local artesian veil)

(mg/liter except pH)

Calcium 3.8 Cyanide 0.005

Magnesium 1.3 Iron 0.02

Potassium 1.05 Copper 0.005

Sulfate 0.01 Zinc 0.024

Nitrate 2.77 Cadmium 0.006

Nitrite 0.05 Chromium 0.05

Ammonia 0.06 Lead 0.001

Phenol 0.001 Alkalinity 63.1

Chlorine 0.001 Hardness 10.6

Chloride 1.90 pH 6.35 units

Fluoride 0.01

68 APPENDIX B: WEIGHTS AND SURVIVAL

69 Table Xllla. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Male Rats

Time Body Weigh t Body Weight Body Weight Interval Mean St d Dev. Surv. Mean Std Dev. Surv. Mean Std Dev. Surv. (weeks) (g) (g) (R) (g) (g) (g)

GrouD 1 ( 0 mg/kg/day) Group 2 (562 mg/kg/day) Group 3 (1000 mg/kg/day)

0 230 19.4 5/5 230 21.4 5/5 231 21.2 5/5 1 263 21.0 5/5 255 34.5 5/5 259 27.8 5/5 2 295 19.2 5/5 284 40.4 5/5 294 29.8 5/5 3 323 29.4 5/5 319 39.9 5/5 327 30.6 5/5 4 358 26.9 5/5 344 36.7 5/5 359 33.9 5/5 5 373 26.3 5/5 357 36.7 5/5 374 33.1 5/5 6 384 27.2 5/5 362 39.4 5/5 376 31.3 5/5 7 406 28.7 5/5 384 42.0 5/5 406 33.9 5/5 8 417 22.1 5/5 400 39.2 5/5 418 34.1 5/5 Mean av. wt gain (g) 18 7 170 187 o % of control wt gain — 90.9 100

Group 4 (173 0 mg/kg/day) Group 5 (3160 mg/kg/day) Group 6 (5620 mg/kg/day)

0 230 21.0 5/5 230 18.4 5/5 228 20.5 5/5 1 246 14.4 5/5 218 18.8 5/5 193 32.6 3/5 2 278 19.4 5/5 240 23.4 5/5 226 — 1/5 3 310 21.1 5/5 273 30.0 5/5 242 - 1/5 4 342 27.1 5/5 299 34.9 5/5 279 - 1/5 5 355 33.2 5/5 298 40.0 5/5 -- 0/5 6 364 36.0 5/5 304 43.2 5/5 7 388 37.5 5/5 344 45.7 5/5 8 404 37.6 5/5 370 48.4 5/5 Mean av. wt gain (g) 17 4 140 51 % of control wt gain 9 3 74.9 27.3 Table Xlllb. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Female Rats

Time Body Weight Body Weight Body Weight Interval Mean Std Dev. Surv. Mean Std Dev. Surv. Mean Std Dev. Surv. (weeks) (g) (g) (g) (g) (g) (g)

Group 1 (0 mg/kg/day) Group 2 (562 mg/kg/day) Group 3 (1000 mg/kg/day)

0 168 14.1 5/5 167 10.9 5/5 170 10.6 5/5 1 183 12.6 5/5 178 7.8 5/5 182 12.1 5/5 2 193 26.0 5/5 195 8.8 5/5 197 12.0 5/5 3 218 29.6 5/5 214 9.1 5/5 218 12.7 5/5 4 235 28.5 5/5 230 10.2 5/5 237 16.8 5/5 5 244 29.8 5/5 236 12.1 5/5 238 18.0 5/5 6 249 31.5 5/5 234 12.1 5/5 239 17.5 5/5 7 266 31.6 5/5 248 9.3 5/5 252 18.9 5/5 8 276 28.9 5/5 258 10.5 5/5 260 20.3 5/5 Mean av. wt gain (g) 108 91 90 % of control wt gain — 84.3 83.3

Group 4 (1730 mg/kg/day) Group 5 (3160 mg/kg/day) Group 6 (5620 mg/kg/day)

0 170 8.3 5/5 170 8.8 5/5 169 14.0 5/5 1 111 9.2 5/5 171 13.3 5/5 178 9.a 2/5 2 183 7.1 5/5 173 19.4 5/5 169 — 1/5 3 202 9.7 5/5 188 24.7 5/5 184 - 1/5 4 227 9.5 5/5 217 23.3 5/5 204 - 1/5 5 226 7.5 5/5 212 21.4 5/5 - - 0/5 6 223 8.0 5/5 217 19.9 5/5 7 239 11.2 5/5 229 16.0 5/5 8 245 14.5 5/5 241 21.7 5/5 Mean av. wt gain (g) 75 71 35 % of control wt gain 69.4 65.7 32.3 Table XlVa. Mean Body Weights and Survival - Trichloroethylene Subchronic Study — Male Mice

Time Body Weight Body Weight Body Weight Interval Mean Std Dev. Surv. Mean Std Dev. Surv. Mean Std Dev. Surv. (weeks) (g) (g) (g) (g) (g)

Group 1 (0 mg/kg/day) Group 2 (1000 mg/kg/day) Group 3 (1780 mg/kg/day)

0 15 0.9 5/5 15 0.9 5/5 15 0.9 5/5 1 16 0.3 5/5 15 1.4 5/5 16 1.7 5/5 2 18 0.5 5/5 19 1.5 5/5 18 0.8 5/5 3 19 0.3 5/5 20 1.5 5/5 19 0.9 5/5 4 20 0.1 5/5 21 1.4 5/5 20 1.2 5/5 5 20 0.5 5/5 21 1.4 5/5 21 1.3 5/5 6 20 0.4 5/5 22 1.2 5/5 21 1.7 5/5 7 20 0.4 5/5 22 0.9 5/5 21 1.2 5/5 8 21 2.1 5/5 23 1.1 5/5 21 1.5 5/5 Mean av. wt gain (g) 6 8 6 % of control wt gain — 133 100

Group 4 (3160 mg/kg/day) Group 5 (5620 mg/kg/day) Group 6 (10,000 mg/kg/day)

0 15 0.9 5/5 15 1.0 5/5 15 1.0 5/5 1 16 1.0 5/5 14 0.3 2/5 -- 0/5 2 18 0.7 5/5 13 3.5 2/5 3 20 1.0 5/5 18 0.5 2/5 4 21 1.3 5/5 18 1.3 2/5 5 22 1.3 5/5 20 1.4 2/5 6 22 1.5 5/5 20 - 1/5 7 22 1.4 5/5 20 - 1/5 8 23 1.2 5/5 20 - 1/5 Mean av. wt gain (g) 8 5 ­ % of control wt gain 133 83.3 Table XlVb. Mean Body Weights and Survival - Trichloroethylene Subchronic Study - Female Mice

Time Body Weight Body Weight Body Weight Interval Mean Std Dev. Surv. Mean Std Dev. Surv. Mean Std Dev, Surv. (weeks) 1E) (g) (g) (g) (g) (g)

Group 1 (0 mg/kg/day) Group 2 (1000 mg/kg/day) Group 3 (1780 mg/kg/day)

0 11 2.2 5/5 11 2.2 5/5 11 2.2 5/5 1 13 1.6 5/5 11 1.8 5/5 13 1.6 5/5 2 16 1.6 5/5 15 1.1 5/5 14 1.0 5/5 3 17 1.0 5/5 17 1.2 5/5 16 1.1 5/5 4 19 0.6 5/5 18 1.1 5/5 18 0.9 5/5 5 19 0.9 5/5 18 1.4 5/5 18 0.5 5/5 6 20 0.9 5/5 19 1.4 5/5 19 1.0 5/5 7 20 0.6 5/5 18 1.6 5/5 19 0.9 5/5 8 21 0.8 5/5 19 2.0 5/5 20 0.9 5/5 Mean av. wt gain (g) 10 8 9 % of control wt gain — 80.0 90.0

Group 4 (3160 rng/kg/day) Group 5 (5620 mg/kg/day) Group 6 (10,000 ιng/kg/day )

0 11 2.4 5/5 11 2.5 5/5 11 2.6 5/5 1 13 1.5 3/5 13 0.5 2/5 -- 0/5 2 15 0.5 3/5 13 4.0 2/5 3 17 1.7 3/5 13 6.9 2/5 4 20 1.5 3/5 20 — 1/5 5 20 1.7 3/5 20 — 1/5 6 20 1.5 3/5 20 — 1/5 7 21 1.3 3/5 20 — 1/5 8 21 1.0 3/5 20 - 1/5 Mean av. wt gain (g) 10 9 ­ % of control wt gain 100 90.0 Table XVa. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Male Rats

Vehicle Controls Low Dose High Dose

Time Body Weighta No. of Body Weight No. of Body Weight No. of Interval Mean Std Dev. Foodb Animals Mean Std Dev Food Animals Mean Std Dev Food Animals (weeks) (g) (g) (g) Weighed (g) (g) (g) Weighed (g) (g) (g) Weighed

0 193 15.0 0 20 193 15.8 0 50 194 16.7 0 50 1 242 18.7 139 20 240 21.8 145 50 237 22.7 128 50 2 288 20.9 155 20 281 26.4 162 50 269 26.5 157 49 3 312 22.2 162 20 309 21.2 166 50 296 27.9 148 48 4 334 22.6 164 20 329 23.5 164 50 313 34.2 156 48 5 360 24.0 161 20 352 25.4 166 50 340 26.2 161 47 6 387 35.2 162 20 377 27.8 168 50 360 28.7 163 47 7 402 27.6 189 20 382 28.8 196 50 375 27.5 170 46 8 412 31.8 157 20 399 30.6 158 50 381 27.8 152 46 9 437 32.8 155 20 423 32.5 157 50 406 31.2 139 46 10 458 34.2 154 20 433 34.7 162 50 406 32.2 156 46 14 504 39.4 153 20 474 40.4 150 50 446 45.3 140 45 18 535 38.9 161 20 503 41.7 163 48 481 42.5 152 44 22 552 38.0 161 20 519 46.3 158 48 493 43.9 148 43 26 570 43.3 156 20 533 47.1 158 47 500 47.2 150 43 30 587 41.5 155 20 544 48.8 164 46 503 51.3 159 42 34 607 40.3 157 20 564 49.3 159 45 528 49.3 148 40 38 617 46.6 153 20 566 50.5 155 45 535 52.1 143 39 42 612 45.2 175 20 565 47.0 177 44 528 57.2 178 38 46 622 47.7 158 20 575 49.7 151 43 526 52.1 151 36 50 618 64.5 152 20 582 49.0 153 42 528 51.3 142 34 54 616 50.2 149 20 573 47.6 147 40 513 48.3 134 30 58 618 45.2 164 20 576 53.5 158 40 508 52.4 137 27 62 628 49.6 167 20 586 47.5 161 38 507 56.7 165 25 66 615 53.6 156 20 562 46.8 156 37 493 57.2 146 19 70 611 59.8 139 19 567 60.8 139 35 506 52.4 137 17 74 581 65.8 151 18 539 62.7 153 33 485 45.8 151 14 78 559 76.7 153 16 523 67.3 158 31 462 47.2 148 12 82 519 92.4 144 15 503 78.3 159 29 450 55.6 169 11 Table XVa. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Male Rats (continued)

86 500 106.7 136 13 490 88.6 152 24 415 52.2 157 10 90 517 94.6 114 9 482 84.5 124 20 422 53.0 120 9 94 515 69.5 138 8 467 96.3 129 18 415 73.7 136 8 98 459 80.7 120 7 449 97.2 144 16 403 64.7 149 7 102 423 42.2 137 4 420 98.6 141 15 389 72.5 141 6 106 394 37.6 94 3 401 88.9 109 11 418 46.9 142 4 110 382 26.9 91 2 383 101.5 114 8 423 45.3 255 3 aCalculated using individual animal weight. ^Average weight per animal per week. Table XVb. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Female Rats

Vehicle Controls Low Dose High Dose

Time Body Weighta No. of Body Weight No. of Body Weight No. of Interval Mean Std Dev.. Foodb Animals Mean Std Dev Food Animals Mean Std Dev Food Animals (weeks) (g) (g) (g) Weighed (g) (g) (g) Weighed (g) (g) (g) Weighed

0 146 11.4 0 20 144 11.0 0 50 144 9.5 0 50 1 169 15.9 110 20 170 13.4 96 50 169 11.7 99 50 2 201 16.1 no 20 183 19.5 122 48 177 21.1 117 50 3 205 14.1 131 20 192 23.7 113 47 196 20.7 110 49 4 216 16.3 133 20 209 18.7 117 46 208 19.9 117 49 5 228 20.0 132 20 220 18.6 127 45 217 22.1 127 49 6 241 25.1 125 20 224 19.2 122 45 226 19.7 118 48 7 242 24.8 153 20 234 19.7 132 44 235 19.8 128 48 8 255 27.9 119 20 240 20.3 113 44 236 18.7 108 48 9 268 26.8 132 20 256 22.0 112 44 251 20.7 112 48 10 280 30.8 130 20 250 21.4 128 44 247 21.0 117 47 14 302 33.8 107 20 271 24.4 98 44 262 26.3 104 47 18 321 37.9 122 20 283 27.9 113 41 276 27.9 112 47 22 330 36.8 119 20 286 31.0 102 37 281 26.5 102 46 26 351 37.6 124 19 293 28.8 110 34 286 30.8 106 45 30 367 39.5 124 19 303 33.9 95 32 304 39.0 93 43 34 382 34.8 128 19 309 33.8 110 31 229 36.3 105 42 38 383 42.7 121 19 309 34.9 104 30 302 38.5 102 41 42 378 52.2 151 19 314 30.7 127 28 306 49.2 130 41 46 382 45.9 127 19 307 30.4 116 28 301 37.1 104 39 50 390 50.9 126 17 312 30.0 114 28 300 42.0 126 39 54 388 51.3 128 17 315 29.8 120 28 307 38.6 113 37 58 396 47.3 135 17 318 34.3 118 27 310 39.7 109 35 62 404 57.4 134 17 318 34.5 129 25 310 39.8 136 34 66 390 54.3 135 17 311 35.7 118 25 304 40.7 116 32 70 399 59.7 116 16 322 46.1 113 23 313 46.1 108 29 74 385 60.4 140 16 303 42.7 127 23 300 45.6 118 24 78 373 58.2 146 16 317 39.9 145 20 317 43.5 135 23 82 382 47.9 143 15 315 43.2 142 20 315 48.1 136 22 86 378 43.4 137 15 311 43.2 131 20 317 51.9 131 22 Table XVb. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Female Rats (continued)

90 364 41.6 101 14 314 33.6 103 19 324 48.9 107 20 94 366 47.7 134 14 313 37.6 123 19 326 55.7 127 20 98 327 61.7 120 13 314 58.3 119 16 321 60.3 129 18 102 336 63.4 134 9 314 66.4 132 15 299 74.3 143 16 106 332 74.9 105 9 308 73.7 114 13 317 70.3 130 13 110 326 80.1 119 8 311 86.0 114 13 311 67.7 136 13 aL,aicuiatea using individual animal i bAverage weight per animal per week. Table XVIa. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Male Mice

Vehicle Controls Low Dose High Dose

Time Body Weighta No. of Body Weight No. of Body Weight No. of Interval Mean Std Dev,. Foodb Animals Mean Std Dev Food Animals Mean Std Dev Food Animals (weeks) (g) (g) (g) Weighed (g) (g) (g) Weighed (g) (g) (g) Weighed

0 17 0.5 0 20 17 2.0 0 50 17 1.1 0 50 1 20 0.0 24 20 19 1.2 23 50 20 0.5 22 50 2 20 1.2 25 20 21 1.0 26 50 21 0.5 25 50 3 22 0.4 22 20 22 0.9 24 50 22 0.9 25 50 4 23 0.1 22 20 23 0.7 24 50 23 0.7 25 50 5 25 0.4 22 20 24 0.7 25 50 25 0.6 25 50 6 25 0.2 22 20 24 0.5 25 50 24 0.5 24 50 7 25 0.4 25 20 26 0.7 27 50 26 1.1 27 50 8 27 0.3 23 20 26 0.8 26 50 26 0.3 26 50

00 9 24 1.0 26 20 26 0.8 28 50 26 0.4 29 50 10 27 0.4 22 20 27 1.2 26 50 28 0.4 25 50 14 28 0.1 23 20 28 0.8 27 50 29 0.4 26 49 18 29 1.7 20 20 30 0.9 24 49 31 0.3 23 46 22 30 0.4 28 20 30 1.5 29 48 30 0.8 29 46 26 31 0.2 24 20 31 0.9 28 48 32 0.7 27 42 30 33 0.1 26 20 32 1.0 28 48 32 0.6 28 38 34 34 0.5 26 19 34 1.2 29 47 34 1.1 31 36 38 34 0.5 24 19 35 1.4 29 47 35 0.5 29 35 42 33 0.1 23 18 33 1.1 27 47 35 0.9 30 35 46 34 0.4 26 18 34 0.9 28 47 35 1.6 30 34 50 34 0.5 23 18 34 1.5 27 47 35 0.7 30 34 54 32 0.4 21 18 33 1.0 26 45 34 0.4 30 33 58 33 1.3 27 18 34 1.1 28 44 35 0.6 33 33 62 35 0.5 27 16 34 0.9 30 44 35 0.2 34 • 30 66 33 0.5 22 15 34 1.5 27 41 34 0.5 32 30 70 33 0.9 31 13 35 1.1 33 41 35 0.5 36 29 74 32 2.7 27 12 34 0.4 34 41 35 0.6 36 27 78 34 0.6 24 8 34 0.8 32 40 35 1.0 39 24 Table XVIa. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Male Mice (continued)

82 33 0.2 33 8 33 0.4 31 38 34 1.2 42 22 86 32 0.1 32 8 32 0.3 32 38 32 1.9 41 22 90 34 0.7 34 8 33 0.7 32 35 34 1.3 38 20 aCalculated using individual animal weight. ^Average weight per animal per week. Table XVIb. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Female Mice

Vehicle Controls Low Dose High Dose

Time Body Weighta No. of Body Weight No. of Body Weight No. of Interval Mean Std Dev. Foodb Animals Mean Std Dev Food Animals Mean Std Dev . Food Animals (weeks) (g) (g) (8) Weighed (g) (g) (g) Weighed (g) (g) (g) Weighed

0 14 0.0 0 20 14 0.6 0 50 14 0.7 0 50 1 17 0.0 22 20 18 0.4 24 50 17 0.6 23 50 2 18 0.3 28 20 18 0.5 28 50 18 0.8 27 50 3 19 0.5 24 20 19 0.3 24 50 19 0.6 23 50 4 19 0.5 30 20 19 0.7 24 50 19 0.7 23 50 5 20 0.5 25 20 20 0.6 24 50 20 0.7 22 50 6 20 0.1 25 20 20 0.3 24 50 19 0.7 23 50 7 21 0.2 32 20 21 0.3 26 50 21 0.5 24 50 8 22 0.9 31 20 21 0.3 27 50 22 0.2 26 50 9 20 0.3 32 20 21 0.7 27 50 21 0.6 26 50 00 10 22 0.4 23 20 22 0.4 23 50 22 0.6 23 50 o 14 23 0.4 27 20 23 0.3 23 49 24 0.5 24 49 18 24 0.1 18 20 25 0.9 21 49 25 1.5 20 49 22 25 0.1 27 19 25 0.6 25 49 24 0.6 24 48 26 25 0.2 22 19 26 0.7 23 49 25 0.5 24 47 30 26 0.1 20 19 26 0.4 23 49 26 0.5 24 47 34 27 0.1 24 19 27 0.4 23 48 27 0.4 24 44 38 27 0.6 19 18 27 0.4 22 46 27 0.4 23 43 42 28 0.8 21 18 28 0.5 23 45 27 0.4 35 41 46 28 0.5 21 18 29 0.5 22 45 27 0.3 22 41 50 28 1.1 20 18 28 0.4 21 45 27 0.6 22 41 54 28 1.1 19 18 27 0.5 21 45 26 0.3 22 41 58 29 0.2 22 18 28 0.5 33 44 27 0.4 23 41 62 28 0.5 25 18 28 0.2 25 44 26 0.4 23 41 66 28 0.9 20 18 28 0.7 22 42 27 0.6 21 41 70 29 0.7 23 18 28 0.6 24 42 28 0.7 23 40 74 29 0.6 21 18 29 0.6 24 42 28 0.7 25 40 78 29 0.6 23 18 29 0.7 25 41 28 0.4 26 40 Table XVIb. Mean Body Weights, Food Consumption, and Survival - Trichloroethylene Chronic Study - Female Mice (continued)

82 28 0.5 24 18 28 0.6 25 40 27 0.6 27 40 86 26 0.0 24 17 27 0.6 25 40 25 1.0 25 40 90 28 0.6 28 17 30 0.6 27 40 28 0.7 26 39

aCalculated using individual animal weight. ^Average weight per animal per week.

00 40 60 80 TIME ON STUDY (WEEKS) Figure 13a. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Male Rats

82 D CONTROL Ο LOW DOSE Δ HIGH DOSE

TIME ON STUDY (WEEKS)

Figure 13b. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Female Rats

83 10 40 60 80 TIME ON STUDY (WEEKS) Figure 14a. Mea n Body Weight vs. Time on Study ­ Trichloroethylene-Treated Male Mice

84 40 60 TIME ON STUDY (WEEKS) Figure 14b. Mean Body Weight vs. Time on Study ­ Trichloroethylene-Treated Female Mice

85

APPENDIX C: STATISTICS

87 Organization of Chronic Data for Statistical Analysis

Statistical analysis begins with examination of the data on survival and pathology of animals in the chronic experiment. These data are presented in detail in the pathology tables in Appendix D. These tables are summarized in this appendix (Tables XVII, XVIIIa, XVIIIb, XlXa, and XlXb). Each animal is listed in its group, identified by its animal number, and arranged in order of the week on study in which it died or was killed or lost. The first column is labeled "Week on Study". An asterisk after the week indicates that the animal's observed lifetime was censored by scheduled or terminal sacrifice, accidental killing, or otherwise lost. Absence of the asterisk indicates that the animal was found dead or was killed while moribund. When mice that were scheduled to be terminally sacrificed at 90 weeks were in fact killed at 91 weeks, 90 weeks is adopted as the week on study of death, so they can be compared with control mice killed at 90 weeks. The second column lists the animal number for reference to the pathology tables. The final column, labeled "Mark", contains letters assigned by the pathologist and the biostatistician in cooperation to sets of pathological diagnoses solely for statistical analysis. The choice of letter is arbitrary, except that more frequent diagnostic sets tend to be assigned to letters early in the alphabet. Each animal is marked if its pathology is included in the set of diagnoses associated with the mark. An animal may have several marks. If the animal has not been evaluated histopathologically, a hyphen appears in the column. A blank space in the column indicates that the animal has been evaluated histopathologically, but has not been marked.

Estimation of Survival Probabilities

The probability that an animal survived each week of the chronic experiment is estimated for each group by the product-limit procedure of Kaplan and Meier (1958). These estimates are listed in Tables XXa, XXb, XXIa, and XXIb in Appendix C. The estimates are also graphed in Figures 2a, 2b, 3a, and 3b. A description of the estimate follows, including a description of the survival tables.

In the survival tables, the first column, labeled j, is the week on study at which death or censoring occurs. The second column, labeled n, is the number of animals at the beginning of week j. The third column, labeled n', is the number of animals alive at the end of week j. The estimate of the conditional probability of the animal surviving week j , given that it was alive at the beginning of the week, is n'/n. If an animal dies during the week, n'/n is less than one. The number of animals dying during the week is n minus n' . Animals whose lifetimes are censored are removed from the set of surviving animals just before the beginning of the week. This removal does not decrease the estimated conditional probability of survival. The number of animals thus removed is found by subtracting the n of this week from the n' of the previously listed week. The fourth column, labeled P, is the product of the factors n'/n for all weeks up to and including the current week j . It is the Kaplan-Meier product-limit esti­ mate of the unconditional probability of an animal surviving from the beginning of the chronic experiment through the end of the current week j.

88 Comparison of Survival Among Groups

A statistical test described by Cox (1972, p. 197), and developed by Mantel (1963, 1966) and Cochran (1954) is used to compare the survival of 2 groups of animals. Table XXc shows results of the test for rats, and Table XXIc shows them for mice. The test accumulates over weeks the observed number of deaths in the higher dosed group minus its expected value under the null hypothesis of equal probability of death in each group. This accumulated statistic is denoted by U in the tables. The test also accumulates the variance of the observed number of deaths. The total variance is denoted by V. A statistic Ζ is computed by dividing U by the square root of V. Since Ζ is distributed approximately as a standard normal random variable, the probability of exceeding Ζ is listed in the column labeled P. Small values of Ρ indicate that the probability of death in the higher dosed group is significantly larger than in the low dosed group at significance level P, indicating longer life for the lower dosed group. The calculation of U and V begins with forming a two-by-two table for each week in which a death has occurred in either group. A typical table is:

Lower Dose Higher Dose Total

Deaths during week Μχ Μ Survivors of week No-Mo Νχ-Μχ N-Μ Animals at risk during week Ν N 0 Nl

The observed number of deaths in the higher dosed group is Μχ, and its expected value under the null hypothesis is Εχ=ΝΜχ/Ν. Their difference, Μχ-Εχ, is accumulated over weeks to form the test statistic U. The variance V of the test statistic U is calculated by accumulating a con­ tribution from each table of (Μ(Ν-Μ)Αχ(Ι-Αχ) / (N-l) , where Αχ=Νχ/Ν. Four comparisons are made by this test: both dosed groups pooled vs. control, low dose vs. control, high dose vs. control, and high dose vs. low dose.

A dose-response table developed by Tarone (1975) is also applied to the two-by-three table:

Control Low Dose High Dose Total

Deaths during week Μ Mo Μχ M 2 Survivors of week NQ-MQ N-Μ Νχ-Μχ N 2 -M2 Animals at risk during week NQ Ni N 2 Ν Dosage do *1 d2

The test statistic U and its variance V are accumulated over weeks. The contribution to U from each table is:

do (M o-E o )+d 1(M 1-E 1 )+d 2(M 2-E 2 ), where Ej=MNj/N for j = 0, 1, or 2.

The contribution to V from each table is:

2 2 2 ((d0 A 0+d 1A 1+d 1A 2 )-(d 0A 0+d 1A 1+d 2A 2 ) )(M(N-M)/N-l)), where Aj=Nj/N, for j=0, 1, or 2.

89 Estimation of Probabilities of Observing Tumors

The probabilities of observing tumors are estimated by a modification of the product-limit procedure of Kaplan and Meier (1958). This modified estimate is described by Saffiotti ejt al. (1972). When an animal dies or is killed, it is evaluated histopathologically and marked as observed to have developed the pathology associated with the mark under study or as not yet having developed it. The animal being marked in this context is analogous to the animal having died in the survival context, and the animal not being marked is analogous to having the lifetime of the animal censored by scheduled sacrifice or loss. If histopathological evaluation of the pathology associated with the mark was not performed, the animal is not considered in the analysis of the mark under study. The probability of survival estimated by the product-limit procedure in the survival context corresponds to the probability of not yet having observed a tumor. A more interesting quantity is the probability of having observed a tumor, which is found by subtracting the probability of not having observed a tumor from one. The estimated probabilities of having observed hepatocellular carcinoma of the liver (mark a) for male mice are given in Table XII, and graphed in Figure 11. Figure 12 shows a comparison of incidence of hepatocellular carcinoma in male and female mice.

Comparison of Probabilities of Observing Tumors Among Groups, Adjusting for Age

The statistical tests of Cox (1972) and Tarone (1975) used to compare survival among groups can be modified to compare the probabilities of observing tumors among groups by employing the analogies described above between death in the survival context and observation of a tumor in the context of observing pathology. Specifically, in the two-by-two and two­ by-three tables of the section on the comparison of survival among groups, "Deaths during week" is replaced by "animals with observed tumor during week". Tumors cannot be observed while the animals are alive. No distinction is made between the natural death of an animal and the censor­ ing of its lifetime in comparisons of tumor probabilities. Animals in which tumors were not observed at histopathological examination are censored from the group of surviving animals for the next week's comparison.

Calculation and interpretation of the Cox and Tarone tests proceed just as in their use in the survival context. Both tests are one-tailed in the direction of increasing probability of tumor with increasing dose. Small values of P indicate significantly greater probability of tumor in higher dosed animals.

These tests compare animals at the same ages in the groups under comparison, removing biases introduced by differing death rates in control and dosed groups.

Results of these tests are shown in Tables Xllla-e for rats and XXVIa-e for mice.

90 Comparison of Probabilities of Observing Tumors Among Groups, by Exact Tests (Not Adjusted for Age)

When the ages of animals at death or sacrifice are ignored, comparison of probabilities of tumors between two groups is performed by the well known Fisher-Irwin exact test, which is a one-tailed test. (See, e_.£., Armitage (1971) for a description.) For three groups, with dosages associated with each group, the test, essentially due to Armitage (1971), employs the linear contrast statistic

U = d0M0 + d1M1 + d2M2 where di = ith dosage and Mi = number of animals with the tumor in the ith group.

The exact distribution of U is computed under the null hypothesis that all groups have the same probability of tumor. The probability P that the observed U is equalled or exceeded is given in tables with the observed proportions and percentages of tumors.

To test for overall heterogeneity among three groups not necessarily related to the dosages, the two degree of freedom chi-square statistic for the classical Pearson test for independence is computed from the data and compared with the exact distribution under the null hypothesis as above. Finally, chi-square statistics with one degree of freedom each are used in exact tests partitioning the two degree of freedom statistic into components for a linear trend on a logistic scale and deviation from the linear trend.

Results of these tests are shown in Tables XXIVa-d for rats and Table XXVII for mice.

91 Table XVII. Identity of Tumor Marks

Mark Tumor

a Hepatocellular carcinoma b Reticulum-cell sarcoma, lymphosarcoma, or malignant lymphoma c Carcinoma or adenocarcinoma of the lung or alveoli d Adenoma of the lung f Aortic body tumor g Fibroadenoma of the mammary glands h Hemangiosarcoma of any site i Malignant giant-cell tumor of soft tissues or fibrosarcoma of the skin or subcutis j Malignant mixed tumor of the kidney k Hamartoma of the kidney 1 Follicular adenoma of the thyroid m Tubular adenocarcinoma of the kidney n Fibroma of the subcutis p Follicular adenocarcinoma of the thyroid q Squamous cell carcinoma of the subcutis r Pilomatrixoma of the skin t Chromophobe adenoma of the pituitary u Adenocarcinoma of the mammary glands v Granulosa-cell carcinoma of the ovaries w Adrenal cortical carcinoma x Liposarcoma of any site y Sarcoma of the endometrium or fibrosarcoma of the uterus z Adenoma of the Harderian gland A Neurofibroma of any site B Adenoma of the kidney C Papilloma of the stomach D Osteosarcoma E Adenocarcinoma of the endometrium F Cystadenoma of the ovaries

92 Table XVIIIa. Data for Statistical Analysis - Trichloroethylene- Treated Male Rats Control Low Dose High Dose Week on Animal Mark Week on Animal Mark Week on Animal Hark Study Number Study Number Study Number 67 3 16 28 2 12 70 11 h 17 36 2 32 76 14 23 10 5 14 76 15 27 6 6 4 82 9 34 35 12 15 82 13 40 30 17 22 83 18 42 34 21 6 87 16 48 3 27 2 88 5 50 7 31 25 88 12 53 41 33 24 90 2 i 60 46 35 40 91 4 61 39 40 33 96 19 jl 65 19 42 39 98 20 67 32 44 3 99 10 jk 67 44 48 18 102 17 72 40 49 30 103 8 74 47 52 5 110* 1 76 18 52 41 110* 6 76 23 1 53 37 110 7 c 80 26 h 54 13 80 33 54 17 82 5 56 27 * Animal's observed 83 38 58 47 lifetime censored 83 43 59 9 by scheduled sacri­ 85 1 61 29 fice, accidental 86 14 62 7 killing or loss. 86 20 62 38 Absence of * means 87 29 65 11 ρ natural death or 88 12 65 28 moribund sacrifice. 90 15 m 65 45 94 25 η 66 49 94 49 68 10 - Histopathology not 96 21 70 20 performed. 97 16 71 16 102 9 72 34 r 103 13 72 50 103 27 75 44 103 31 76 21 104 37 82 19 f 107 8 83 43 107 45 88 31 108 4 i 91 36 h 110* 2 kp 97 42 110* 11 q 99 8 110* 17 102 35 110* 22 103 1 110* 24 109 48 110 42 110* 23 110* 48 110* 26 h 110* 50 110* 46

93 Table XVIIIb. Data foi: Statistical Analysis - Tnchloroethylene- Treated Female Rats Control Low Dose Iiigh Dose Week on Animal Mark Week on Animal Mark Week on Animal Mark Study Number Study Number Study Number 25 8 2 24 3 13 47 16 2* 29 — 5 2 47 20 2 42 9 32 68 6 t 3 2 h 21 36 79 1 5 20 24 49 87 7 7 47 28 8 97 11 g 15 1 28 16 98 15 16 30 30 43 99 16 38 35 40 102 14 tu 19 11 43 38 104 19 t 21 12 46 45 108 12 21 14 50 4 110* 3 gt 21* 21 _ 53 7 110* 4 bv 22 5 54 41 110* 5 22 39 58 24 110* 9 25 17 59 17 110* 10 g 26 36 63 31 110* 13 28 35 64 22 110* 17 33 33 67 28 110* 18 34 19 69 12 40 45 69 27 42 22 70 18 * Animal's observed 57 6 70 20 lifetime censored 60 10 72 14 by scheduled sacri- 61 44 73 1 fice, accidental 68 27 73 42 killing or loss. 69 25 74 39 Absence of * means 75 15 82 26 natural death or 75 16 b 86 48 moribund sacrifice. 77 48 89 47 86 34 95 21 96 23 w 96 50 t - Histopathology not 96 46 101 29 g performed. 98 32 X 101 35 100 13 η 102 19 102 49 103 33 g 104 40 g 104 34 110* 3 110* 3 110* 4 110* 5 t 110* 7 gt 110* 6 b 110* 8 110* 9 gt 110* 9 110* 10 g 110* 18 g 110* 11 110* 26 y 110* 15 110* 28 110* 23 gt 110* 31 t 110* 25 110* 37 110* 30 gp 110* 41 110* 37 gy 110* 43 g 110* 44 110 50 g 110* 46 t

94 Table XIXa. Data for Statistical Analysis - Trichloroethylene- Treated Male Mice Control Low Dose High Dose Week on Animal Mark Week on Animal Mark Week on Animal Mark Study Number Study Number Study Number 32 10 16 10 13 10 39 9 18 50 b 14 40 60 8 31* 9 15 49 61 7 51 30 15* 50 64 6 53 40 24 19 66 20 58 8 24 20 68 5 63 39 25 47 72 19 a 65 20 25 48 76 4 65 38 27 46 a 76 18 b 77 19 28 18 a 77 17 i 81 28 b 28 45 78 3 81 29 ad 29 30 90* 1 i 86 49 a 30 9 90* 2 88 7 ad 30 17 a 90* 11 i 88 18 36 8 a 90* 12 90* 1 ad 42 7 90* 13 90* 2 η 53 39 a 90* 14 90* 3 a 60 5 90* 15 90* 4 abd 60 6 90* 16 90* 5 a 61 16 a 90* 6 a 70 38 a 90* 11 a 71 15 a * Animal's observed 90* 12 a 72 37 lifetime censored 90* 13 a 74 29 a by scheduled sacri­ 90* 14 ζ 75 4 fice, accidental 90* 15 ab 75 36 _ killing or loss. 90* 16 78 44 aA Absence of * means 90* 17 a 83 35 ac natural death or 90* 21 a 88 3 ab moribund sacrifice. 90* 22 90* 1 a 90* 23 90* 2 a 90* 24 a 90* 11 a - Histopathology not 90* 25 90* 12 a performed. 90* 26 a 90* 13 aB 90* 27 90* 14 a 90* 31 a 90* 21 a 90* 32 a 90* 22 a 90* 33 a 90* 23 a 90* 34 a 90* 24 b 90* 35 a 90* 25 a 90* 36 a 90* 26 a 90* 37 ad 90* 27 a 90* 41 a 90* 28 a 90* 42 90* 31 d 90* 43 a 90* 32 a 90* 44 90* 33 aC 90* 45 90 34 ai 90* 46 90* 41 a 90* 47 90* 42 a 90* 48 a 90* 43 ah

95 Table XlXb. Data for Statistical Analysis - Trichloroethylene- Treated Female Mice Control Low Dose High Dose Week on Animal Mark Week on Animal Mar k Wee k on Animal Mar k Study Number Study Number Study Numbe r 20* 10 10 20 14* 10 — 37* 9 32* 10 22* 40 - 83* 8 D 37 19 26* 50 - 90* 1 38 18 32 30 90* 2 41* 9 32 49 90* 3 Ε 55 30 33 39 90* 4 63 29 38 38 90* 5 66 40 39 20 90* 6 76 17 b 40 37 90* 7 d 81 16 b 69 9 b 90* 11 90* 1 b 88 36 b 90* 12 90* 2 90* 1 b 90* 13 90* 3 ay 90* 2 90* 14 b 90* 4 90* 3 90* 15 90* 5 90* 4 90* 16 90* 6 90* 5 90* 17 90* 7 a 90* 6 a 90* 18 90* 8 90* 7 90* 19 90* 11 90* 8 ac 90* 20 90* 12 a 90* 11 90* 13 90* 12 a 90* 14 90* 13 a * Animal's observed 90 * 15 90* 14 lifetime censored 90* 21 90* 15 d by scheduled sacri- 90* 22 90* 16 fice, accidental 90* 23 90* 17 killing or loss. 90* 24 90* 18 b Absence of * means 90* 25 90* 19 natural death or 90* 26 90* 21 moribund sacrifice. 90 * 27 c 90* 22 b 90* 28 90* 23 d 90* 31 90* 24 a - Histopathology not 90* 32 a 90* 25 ac performed. 90* 33 V 90* 26 a 90* 34 90* 27 90* 35 i 90* 28 90* 36 bd 90* 29 90* 37 90* 31 90* 38 u 90* 32 90* 39 90* 33 90* 41 90* 34 d 90* 42 bd 90* 35 90* 43 czF 90* 41 abd 90* 44 90* 42 a 90* 45 90* 43 90* 46 90* 44 a 90* 47 90* 45 90* 48 90* 46 ad 90* 49 90* 47 90* 50 90* 48

96 Table XXa. Product-Limit Estimates of Probability of Survival ­ Trichloroethylene-Treated Male Rats Control Low Dose High Dose j n n' P j n n' P j n n' P 0 50 50 1.000 0 50 50 1 .000 0 20 20 1.000 16 50 49 .980 2 50 48 .960 67 20 19 .950 17 49 48 .960 5 48 47 .940 70 19 18 .900 23 48 47 .940 6 47 46 .920 76 18 16 .800 27 47 46 .920 12 46 45 .900 82 16 14 .700 34 46 45 .900 17 45 44 .880 83 14 13 .650 40 45 44 .880 21 44 43 .860 87 13 12 .600 42 44 43 .860 27 43 42 .840 88 12 10 .500 48 43 42 .840 31 42 41 .820 90 10 9 .450 50 42 41 .820 33 41 40 .800 91 9 8 .400 53 41 40 .800 35 40 39 .780 96 8 7 .350 60 40 39 .780 40 39 38 .760 98 7 6 .300 61 39 38 .760 42 38 37 .740 99 6 5 .250 65 38 37 .740 44 37 36 .720 102 5 4 .200 67 37 35 .700 48 36 35 .700 103 4 3 .150 72 35 34 .680 49 35 34 .680 110 3 2 .100 74 34 33 .660 52 34 32 .640 76 33 31 .620 53 32 31 .620 j - Week on study 80 31 29 .580 54 31 29 .580 82 29 28 .560 56 29 28 .560 n = No, of animals 83 28 26 .520 58 28 27 .540 alive at beginning 85 26 25 .500 59 27 26 .520 of the week 86 25 23 .460 61 26 25 .500 87 23 22 .440 62 25 23 .460 n' = No. of animals 88 22 21 .420 65 23 20 .400 surviving the week 90 21 20 .400 66 20 19 .380 P = Kaplan-Meier 94 20 18 .360 68 19 18 .360 estimate of sur­ 96 18 17 .340 70 18 17 .340 97 17 16 .320 71 17 16 .320 vival probability 102 16 15 .300 72 16 14 .280 103 15 12 .240 75 14 13 .260 104 12 11 .220 76 13 12 .240 107 11 9 .180 82 12 11 .220 108 9 8 .160 83 11 10 .200 110 8 7 .140 88 10 9 .180 91 9 8 .160 97 8 7 .140 99 7 6 .120 102 6 5 .100 103 5 4 .080 109 4 3 .060 110 3 3 .060

97 Table XXb. Product-Limit Estimates of Probability of Survival ­ Trichloroethylene-Treated Female Rats Control Low Dose High Dose

j η η ' Ρ j η η ' Ρ j η η ' Ρ 0 20 20 1 .000 0 50 50 1.000 0 50 50 1 .000 25 20 19 .950 2 50 48 .960 3 50 49 .980 47 19 17 .850 3 47 46 .940 5 49 48 .960 68 17 16 .800 5 46 45 .919 9 48 47 .940 79 16 15 .750 7 45 44 .899 21 47 46 .920 87 15 14 .700 15 44 43 .878 24 46 45 .900 97 14 13 .650 16 43 41 .837 28 45 43 .860 98 13 12 .600 19 41 40 .817 30 43 42 .840 99 12 11 .550 21 40 38 .776 35 42 41 .820 102 11 10 .500 22 37 35 .734 43 41 40 .800 104 10 9 .450 25 35 34 .713 46 40 39 .780 108 9 8 .400 2 6 34 33 .692 50 39 38 .760 110 8 8 .400 2 8 33 32 .671 53 38 37 .740 33 32 31 .650 54 37 36 .720 j = Week on study 34 31 30 .629 58 36 35 .700 40 30 29 .608 59 35 34 .680 n = No. of animals 42 29 28 .587 63 34 33 .660 alive at beginning 5 7 28 27 .566 64 33 32 .640 of the week 60 27 26 .545 67 32 31 .620 61 26 25 .524 69 31 29 .580 n' = No. of animals 68 25 24 .503 70 29 27 .540 surviving the week 6 9 24 23 .482 72 27 26 .520 75 23 21 .441 73 26 24 .480 Ρ = Kaplan-Meier 77 21 20 .420 74 24 23 .460 estimate of sur­ 86 20 19 .399 82 23 22 .440 vival probability 96 19 17 .357 86 22 21 .420 98 17 16 .336 89 21 20 .400 100 16 15 .315 95 20 19 .380 102 15 14 .294 96 19 18 .360 104 14 13 .273 101 18 16 .320 110 13 12 .252 102 16 15 .300 103 15 14 .280 104 14 13 .260 110 13 13 .260

98 Table XXc. Statistical Tests Comparing Estimated Probability of Survival among Control and Trichloroethylene-Treated Rats

Male Rats Female Rats Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 23.60 51.47 3.29 0.001 8.48 50.64 1.19 0.117 Dosed vs. Control 6.47 18.17 1.52 0.064 7.13 14.55 1.87 0.031 Low Dose vs. Control 0.88 12.44 0.25 0.402 6.33 11.08 1.90 0.028 High Dose vs. Control 11.12 15.07 2.86 0.002 5.50 11.04 1.65 0.049 High Dose vs. Low Dose 13.94 19.86 3.13 0.001 -2.56 17.84 -0.61 0.728

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z, See appendix on statistical methodology for full definition.

99 Table XXIa. Product-Limit Estimates of Probability of Survival Trichloroethylene-Treated Male Mice

Control Low Dose High Dose

j η η' Ρ j η n' Ρ j η n' Ρ 0 20 20 1.000 0 50 50 1.000 0 50 50 1.000 32 20 19 .950 16 50 49 .980 13 50 49 .980 39 19 18 .900 18 49 48 .960 14 49 48 .960 60 18 17 .850 31 48 48 .960 15 48 47 .940 61 17 16 .800 51 47 46 .940 24 46 44 .899 64 16 15 .750 53 46 45 .919 25 44 42 .858 66 15 14 .700 58 45 44 .899 27 42 41 .838 68 14 13 .650 63 44 43 .878 28 41 39 .797 72 13 12 .600 65 43 41 .837 29 39 38 .777 76 12 10 .500 77 41 40 .817 30 38 36 .736 77 10 9 .450 81 40 38 .776 36 36 35 .715 78 9 8 .400 86 38 37 .756 42 35 34 .695 90 8 8 .400 88 37 35 .715 53 34 33 .674 90 35 35 .715 60 33 31 .633 j ­ Week on study 61 31 30 .613 70 30 29 .593 η = No. of animals 71 29 28 .572 alive at beginning 72 28 27 .552 of the week 74 27 26 .531 75 26 24 .490 n' == No, of animals 78 24 23 .470 surviving the week 83 23 22 .450 88 22 21 .429 Ρ = Kaplan-Meier 90 21 20 .409 estimate of sur­ vival probability

Table XXIb. Product-Limit Estimates of Probability of Survival Trichloroethylene-Treated Female Mice

Control Low Dose High Dose

j η η' Ρ j η n' Ρ j η n' Ρ 0 20 20 1.000 0 50 50 1 .000 0 50 50 1.000 20 20 20 1.000 10 50 49 .980 14 50 50 1.000 37 19 19 1.000 32 49 49 .980 22 49 49 1.000 83 18 18 1.000 37 48 47 .960 26 48 48 1.000 90 17 17 1.000 38 47 46 .939 32 47 45 .957 41 46 46 .939 33 45 44 .936 j = Week on study 55 45 44 .918 38 44 43 .915 63 44 43 .897 39 43 42 .894 η = No. of animals 66 43 42 .877 40 42 41 .872 alive at beginning 76 42 41 .856 69 41 40 .851 of the week 81 41 40 .835 88 40 39 .830 90 40 40 .835 90 39 39 .830 n' = No. of animals surviving the week Ρ = Kaplan-Meier estimate of sur­ vival probability

100 Table XXIc. Statistical Tests Comparing Estimated Probability of Survival among Control and Trichloroethylene-Treated Mice

Male Mice Female Mice

Comparison U V Z P U V Z P

Dose- Response 6.67 26.11 1.30 0.096 4.29 8.32 1.49 0.068 Dosed vs. Control -3.02 7.42 -1.11 0.866 2.75 2.27 1.82 0.034 Low Dose vs. Control -5.72 4.68 -2.64 0.996 2.32 1.65 1.81 0.035 High Dose vs. Control 1.11 8.77 0.37 0.354 2.36 1.66 1.83 0.033 High Dose vs. Low Dose 10.72 10.35 3.33 0.001 0.21 3.99 0.10 0.459

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Z = U divided by square root V. P = probability that a standard normal random variable is greater than Z, See appendix on statistical methodology for full definition.

101 Table XXII. Tumor Incidence - Trichloroethylene-Treated Rats

Malei Rats Female Rats Low High Low High Control Dose Dose Control Dose Dose

Mark b Reticulum-cell sarcoma, lymphosarcoma, or malignant lymphoma

Before 110 weeks 0/17 0/42 0/47 0/12 1/35 0/37 At 110 weeks 0/3 0/8 0/3 1/8 0/13 1/13 Total 0/20 0/50 0/50 1/20 1/48 1/50

Mark g Fibroadenoma of the mammary glands

Before 110 weeks 0/17 0/42 0/47 1/12 1/35 2/37 At 110 weeks 0/3 0/8 0/3 2/8 4/13 5/13 Total 0/20 0/50 0/50 3/20 5/48 7/50

Mark h Hemangiosarcoma of any site

Before 110 weeks 1/17 1/42 1/47 0/12 1/35 0/37 At 110 weeks 0/3 0/8 1/3 0/8 0/13 0/13 Total 1/20 1/50 2/50 0/20 1/48 0/50

Mark p Follicular adenocarcinoma of the thyroid

Before 110 weeks 0/17 0/42 1/47 0/12 0/35 0/37 At 110 weeks 0/3 1/8 0/3 0/8 0/13 1/13 Total 0/20 1/50 1/50 0/20 0/48 1/50

Mark t Chromophobe adenoma of the pituitary

Before 110 weeks 0/17 0/42 0/47 3/12 0/35 2/37 At 110 weeks 0/3 0/8 0/3 1/8 2/13 4/13 Total 0/20 0/50 0/50 4/20 2/48 6/50

Animals with Tumors (Benign and Malignant)a Before 110 weeks 4/17 5/42 4/47 4/12 6/35 4/37 At 110 weeks 1/3 2/8 1/3 3/8 6/13 8/13 Total 5/20 7/50 5/50 7/20 12/48 12/50

Includes tumors other than those listed above.

102 Table XXIIIa. Statistical Tests Comparing Estimated Probability of Observing Reticulum-cell Sarcoma, Lymphosarcoma, or Malignant Lymphoma (Mark b) Control and Trichloroethylene-Treated Rats

Male Rats Female Rats Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 0.00 0.00 -0.41 1.77 -0.31 0.620 Dosed vs. Control 0.00 0.00 -0.27 0.54 -0.37 0.644 Low Dose vs. Control 0.00 0.00 -0.21 0.48 -0.30 0.619 High Dose vs. Control 0.00 0.00 -0.24 0.45 -0.36 0.639 High Dose vs. Low Dose 0.00 0.00 0.00 0.50 0.00 0.500

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z, See appendix on statistical methodology for full definition.

103 Table XXIIIb. Statistical Tests Comparing Estimated Probability of Observing Fibroadenoma of the Mammary Glands (Mark g) among Control and Trichloroethylene-Treated Rats Male Rats Female Rats Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 0.00 0.00 1.91 7.09 0.72 0.237 Dosed vs. Control 0.00 0.00 0.64 2.16 0.44 0.330 Low Dose vs. Control 0.00 0.00 0.15 1.55 0.12 0.451 High Dose vs. Control 0.00 0.00 0.88 1.88 0.64 0.259 High Dose vs. Low Dose 0.00 0.00 0.94 2.28 0.62 0.267

Table XXIlie. Statistical Tests Comparing Estimated Probability of Observing Hemangioma of Any Site (Mark h) among Control and Trichloroethylene-Treated Rats Male Rats Female Rats Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 1.08 1.89 0.79 0.215 -0.26 0.54 -0.35 0.638 Dosed vs. Control -0.01 0.74 -0.02 0.506 0.17 0.14 0.46 0.324 Low Dose vs. Control -0.31 0.45 -0.46 0.677 0.30 0.21 0.66 0.255 High Dose vs. Control 0.51 0.75 0.59 0.276 0.00 0.00 High Dose vs. Low Dose 1.14 0.61 1.45 0.073 -0.52 0.25 -1.04 0.851

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z, See appendix on statistical methodology for full definition.

104 Table XXIIId. Statistical Tests Comparing Estimated Probability of Observing Follicular Adenocarcinoma of the Thyroid (Mark p) among Control and Trichloroethylene-Treated Rats

Male Rats Female Rats Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 0.96 0.96 0.98 0.163 0.85 0.60 1.10 0.135 Dosed vs. Control 0.46 0.35 0.77 0.219 0.24 0.18 0.55 0.289 Low Dose vs. Control 0.27 0.20 0.61 0.270 0.00 0.00 High Dose vs. Control 0.47 0.25 0.93 0.176 0.38 0.24 0.78 0.216 High Dose vs. Low Dose 0.35 0.43 0.53 0.297 0.50 0.25 1.00 0.159

Table XXIIIe. Statistical Tests Comparing Estimated Probability of Observing Chromophobe Adenoma of the Pituitary (Mark t) among Control and Trichloroethylene-Treated Rats

Male Rats Female Rats

Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 0.00 0.00 0.30 6.54 0.12 0.454 Dosed vs. Control 0.00 0.00 -1.08 1.98 -0.76 0.778 Low Dose vs. Control 0.00 0.00 -1.61 1.36 -1.38 0.916 High Dose vs. Control 0.00 0.00 -0.11 2.14 -0.08 0.531 High Dose vs. Low Dose 0.00 0.00 1.95 1.70 1.50 0.067

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z. See appendix on statistical methodology for full definition.

105 Table XXIVa. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma of the Liver among Pooled Control and Trichloroethylene-Treated Rats

14ale Rats Female Rats Veh. Low High Exact Veh. Low High Exact Cont. Dose Dose Test Cont. Dose Dose Test

Comparison r/n r/n r/n P r/n r/n r/n P

Dose-Response 1/99 0/50 0/50 0/98 0/48 0/50 Veh. Control 1% 0% 0% 1.000 0% 0% 0% 1.000

Dosed vs. 1/99 0/100 0/98 0/98 Veh. Control 1% 0% 1.000 0% 0% 1.000

Low Dose vs. 1/99 0/50 0/98 0/48 Veh. Control 1% 0% 1.000 0% 0% 1.000

High Dose vs. 1/99 0/50 0/98 0/50 Veh. Control 1% 0% 1.000 0% 0% 1.000

High Dose vs. 0/50 0/50 0/48 0/50 Low Dose 0% 0% 1.000 0% 0% 1.000

Comparison Chi-square df P Chi-square df P

Among High Dose, Low Dose and Vehicle Control 1.02 2 1.000 1.00 2 1.00 0 Dose-Response Trend Vehicle Control 0.83 1 0.749 1.00 1 1.00 0 Deviation from Trend Vehicle Control 0.19 1 1.000 0.00 1 1.00 0 r = Number of animals with observed mark. n = Number of animals examined histopathologically for the mark. P = Exact probability of exceeding or equaling observed test statistic under null hypothesis, df = Degrees of freedom. See section on statistical methodology for explanation of tests.

106 Table XXIVb. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Trichloroethylene-Treated Rats

Male Rats ]Female Rats Veh. Low High Exact Veh. Low High Exact Cont. Dose Dose Test Cont. Dose Dose Test

Comparison r/n r/n r/n Ρ r/n r/n r/n Ρ

Dose-Response 1/99 0/50 0/50 2/98 0/48 0/50 Veh. Control 1% 0% 0% 1.000 2% 0% 0% 1.000

Dosed vs. 1/99 0/100 2/98 0/98 Veh. Control 1% 0% 1.000 2% 0% 1.000

Low Dose vs. 1/99 0/50 2/98 0/48 Veh. Control 1% 0% 1.000 2% 0% 1.000

High Dose vs. 1/99 0/50 2/98 0/50 Veh. Control 1% 0% 1.000 2% 0% 1.000

High Dose vs. 0/50 0/50 0/48 0/50 Low Dose 0% 0% 1.000 0% 0% 1.000

Comparison Chi-square df Ρ Chi-square df Ρ

Among High Dose, Low Dose and Vehicle Control 1.02 2 1.000 2.04 2 0.371

Dose-Response Trend Vehicle Control 0.83 1 0.749 1.67 1 0.310

Deviation from Trend Vehicle Control 0.19 1 1.000 0.37 1 1.000 r = Number of animals with observed mark. η = Number of animals examined histopathologically for the mark. Ρ = Exact probability of exceeding or equaling observed test statistic under null hypothesis, df = Degrees of freedom. See section on statistical methodology for explanation of tests.

107 Table XXIVc. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma of the Liver among Pooled Control and Carbon Tetrachloride-Treated Rats

Male Rats Female Rats Veh. Low High Exact Veh. Low High Exact Cont. Dose Dose Test Cont. Dose Dose Test

Comparison r/n r/n r/n Ρ r/n r/n r/n Ρ

Dose-Response 1/99 2/50 2/50 0/98 4/49 1/49 Veh. Control 1% 4% 4% 0.177 0% 8% 2% 0.174

Dosed vs. 1/99 4/100 0/98 5/98 Veh. Control 1% 4% 0.187 0% 5% 0.030

Low Dose vs. 1/99 2/50 0/98 4/49 Veh. Control 1% 4% 0.261 0% 8% 0.011

High Dose vs. 1/99 2/50 0/98 1/49 Veh. Control 1% 4% 0.261 0% 2% 0.333

High Dose vs. 2/50 2/50 4/49 1/49 Low Dose 4% 4% 0.691 8% 2% 0.972

Comparison Chi-square df Ρ Chi-square df Ρ

Among High Dose, Low Dose and Vehicle Control 1.82 2 0.601 8.83 2 0.011

Dose-Response Trend Vehicle Control 1.48 1 0.282 1.51 1 0.281

Deviation from Trend Vehicle Control 0.33 1 0.635 7.31 1 0.014 r = Number of animals with observed mark. η = Number of animals examined histopathologically for the mark. Ρ = Exact probability of exceeding or equaling observed test statistic under null hypothesis, df = Degrees of freedom. See section on statistical methodology for explanation of tests.

108 Table XXIVd. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Carbon Tetrachloride-Treated Rats

Male Rats Female Rats Untr. Veh . Low High Exact Untr. Veh. Low High Exact Cont. Cont. Dose Dose Test Cont. Cont. Dose Dose Test

Comparison r/n r/n r/n r/n Ρ r/n r/n r/n r/n Ρ

Dose-Response 1/99 4/50 3/50 2/98 6/49 4/49 Veh. Control 1% 8% 6% 0.070 2% 12% 8% 0.057

Dose-Response 0/20 4/50 3/50 0/20 6/49 4/49 Untr. Control 0% 8% 6% 0.353 0% 12% 8% 0.325

Dosed vs. 1/99 7/100 2/98 10/98 Veh. Control 1% 7% 0.033 2% 10% 0.016

Dosed vs. 0/20 7/100 0/20 10/98 Untr. Control 0% 7% 0.269 0% 10% 0.144

Low Dose vs. 1/99 4/50 2/98 6/49 Veh. Control 1% 8% 0.044 2% 12% 0.017

Low Dose vs. 0/20 4/50 0/20 6/49 Untr. Control 0% 8% 0.251 0% 12% 0.117

High Dose vs. 1/99 3/50 2/98 4/49 Veh. Control 1% 6% 0.110 2% 8% 0.096

High Dose vs. 0/20 3/50 0/20 4/49 Untr. Control 0% 6% 0.358 0% 8% 0.245

High Dose vs. 4/50 3/50 6/49 4/49 Low Dose 8% 6% 0.782 12% 8% 0.841

Veh. Con. vs. 0/20 1/99 0/20 2/98 Untr. Control 0% 1% 0.832 0% 2% 0.689

(continued)

109 Table XXIVd. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Carbon Tetrachloride-Treated Rats (continued)

Comparison Chi-square df Ρ Chi-square df Ρ

Among High Dose, Low Dose and Vehicle Control 4.89 2 0.100 6.39 2 0.046

Dose-Response Trend Vehicle Control 2.98 1 0.128 3.23 1 0.105

Deviation from Trend Vehicle Control 1.90 1 0.203 3.16 1 0.079

Among High Dose, Low Dose and Untreated Control 1.67 2 0.528 2.76 2 0.259

Dose-Response Trend Untreated Control 0.46 1 0.601 0.50 1 0.511

Deviation from Trend Untreated Control 1.21 1 0.333 2.26 1 0.165

r = Number of animals with observed mark. η = Number of animals examined histopathologically for the mark. Ρ = Exact probability of exceeding or equaling observed test statistic under null hypothesis, df = Degrees of freedom. See section on statistical methodology for explanation of tests.

110 Table XXV. Tumor Incidence - Trichloroethylene-Treated Mice

Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose Mark a Hepatocellular carcinoma of the liver Before 90 weeks 1/12 3/15 12/27 0/3 0/10 0/8 At 90 weeks 0/8 23/35 19/21 0/17 4/40 11/39 Total 1/20 26/50 31/48 0/20 4/50 11/47

•Mark b Reticulum-cell sarcoma, lymphosarcoma, or malignant lymphoma Before 90 weeks 1/12 2/15 1/27 0/3 2/10 2/8 At 90 weeks 0/8 2/35 1/21 1/17 3/40 4/39 Total 1/20 4/50 2/48 1/20 5/50 6/47

Mark c Carcinoma or adenocarcinoma of the lung or alveoli Before 90 weeks 0/12 0/15 1/27 0/3 0/10 0/8 At 90 weeks 0/8 0/35 0/21 0/17 2/40 2/39 Total 0/20 0/50 1/48 0/20 2/50 2/47

Mark d Adenoma of the lung Before 90 weeks 0/12 2/15 0/27 0/3 0/10 0/8 At 90 weeks 0/8 3/35 1/21 1/17 2/40 5/39 Total 0/20 5/50 1/48 1/20 2/50 5/47

Marks c or d Carcinoma, adenocarcinoma,or adenoma of the lung or alveoli Before 90 weeks 0/12 2/15 1/27 0/3 0/10 0/8 At 90 weeks 0/8 3/35 1/21 1/17 4/40 7/39 Total 0/20 5/50 2/48 1/20 4/50 7/47

Animals with Tumors (Benign and Malignant)a Before 90 weeks 3/12 5/15 12/27 1/3 2/10 2/8 At 90 weeks 2/8 25/35 21/21 3/17 12/40 17/39 Total 5/20 30/50 33/48 4/20 14/50 19/47

Includes tumors other than those listed above.

Ill Table XXVIa. Statistical Tests Comparing Estimated Probability of Observing Hepatocellular Carcinoma (Mark a) among Control and Trichloroethylene-Treated Mice Male Mice Female Mice Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 18.57 13.34 5.08 0.000 7.56 6.79 2.90 0.002 Dosed vs. Control 6.72 3.68 3.51 0.000 2.66 1.86 1.95 0.026 Low Dose vs. Control 4.04 2.27 2.68 0.004 1.19 0.79 1.34 0.090 High Dose vs. Control 8.45 4.18 4.13 0.000 3.34 1.90 2.42 0.008 High Dose vs. Low Dose 9.27 6.08 3.76 0.000 3.59 3.08 2.05 0.020

Table XXVIb. Statistical Tests Comparing Estimated Probability of Observing Reticulum-cell Sarcoma, Lymphosarcoma, or Malignant Lymphoma (Mark b) among Control and Trichloroethylene-Treated Mice

Male Mice Female Mice Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response -0.40 2.96 -0.23 0.591 2.26 6.07 0.92 0.180 Dosed vs. Control -0.06 0.80 -0.07 0.527 1.13 1.67 0.88 0.190 Low Dose vs. Control 0.06 0.82 0.06 0.476 0.80 1.21 0.72 0.234 High Dose vs. Control -0.12 0.62 -0.16 0.563 1.12 1.40 0.95 0.172 High Dose vs. Low Dose -0.34 1.39 -0.28 0.612 0.57 2.61 0.35 0.362

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z. See appendix on statistical methodology for full definition.

112 Table XXVIc. Statistical Tests Comparing Estimated Probability of Observing Carcinoma or Adenocarcinoma of the Lung or Alveoli (Mark c) among Control and Trichloroethylene-Treated Mice

Male Mice Female Mice Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 0.78 0.40 1.23 0.109 1.08 2.06 0.76 0.225 Dosed vs. Control 0.12 0.10 0.36 0.358 0.71 0.56 0.94 0.173 Low Dose vs. Control 0.00 0.00 0.60 0.41 0.93 0.176 High Dose vs. Control 0.26 0.19 0.59 0.277 0.61 0.42 0.94 0.173 High Dose vs. Low Dose 0.62 0.23 1.29 0.099 0.03 0.96 0.03 0.490

Table XXVId. Statistical Tests Comparing Estimated Probability of Observing Adenoma of the Lung (Mark d) among Control and Trichloroethylene-Treated Mice

Male Mice Female Mice Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response -0.23 2.37 -0.15 0.560 2.17 3.93 1.09 0.138 Dosed vs. Control 0.73 0.62 0.93 0.177 0.42 1.08 0.40 0.344 Low Dose vs. Control 0.90 0.72 1.07 0.144 -0.11 0.61 -0.14 0.554 High Dose vs. Control 0.28 0.20 0.62 0.268 0.82 1.15 0.76 0.222 High Dose vs. Low Dose -1.24 1.35 -1.06 0.856 1.54 1.62 1.22 0.112

U - observed test statistic minus its expected value under the null hypothesis· V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z. See appendix on statistical methodology for full definition.

113 Table XXVIe. Statistical Tests Comparing Estimated Probability of Observing Carcinoma, Adenocarcinoma, or Adenoma of the Lung or Alveoli (Mark c or d) among Control and Trichloroethylene-Treated Mice

Male Mice Female Mice Comparison U V Ζ Ρ υ V Ζ Ρ Dose- Response 0.55 2.77 0.33 0.370 3.25 5.63 1.37 0.085 Dosed vs. Control 0.85 0.72 1.00 0.160 1.13 1.55 0.90 0.183 Low Dose vs. Control 0.90 0.72 1.07 0.144 0.49 0.97 0.50 0.309 High Dose vs. Control 0.53 0.39 0.85 0.197 1.43 1.48 1.18 0.120 High Dose vs. Low Dose -0.62 1.59 -0.49 0.688 1.57 2.40 1.01 0.156

U = observed test statistic minus its expected value under the null hypothesis. V = variance of U. Ζ = U divided by square root V. Ρ = probability that a standard normal random variable is greater than Z, See appendix on statistical methodology for full definition.

114 Table XXVII. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Trichloroethylene-Treated Mice

Male Mice Female Mice Untr. Veh . Lo w High Exac t Untr. Veh . Lo w High Exac t Cont. Cont . Dos e Dos e Tes t Cont. Cont . Dos e Dos e Tes t

Comparison r/n r/ n r/ n r/ n Ρ r/n r/ n r/ n r/ n Ρ

Dose-Response 5/77 26/5 0 31/4 8 1/80 4/5 0 11/4 7 Veh. Control 6% 52% 65% 0.00 0 1% 8% 23% 0.00 0

Dose-Response 5/70 26/50 31/4 8 2/76 4/50 11/4 7 Untr. Control 7% 52% 65% 0.00 0 3% 8% 23% 0.00 0

Dosed vs. 5/77 57/98 1/80 15/97 Veh. Control 6% 58% 0.000 1% 15% 0.001

Dosed vs. 5/70 57/98 2/76 15/97 Untr. Control 7% 58% 0.000 3% 15% 0.004

Low Dose vs. 5/77 26/5 0 1/80 4/5 0 Veh. Control 6% 52% 0.000 1% 8% 0.072

Low Dose vs. 5/70 26/50 2/76 4/50 Untr. Control 7% 52% 0.000 3% 8% 0.169

High Dose vs. 5/77 31/48 1/80 11/47 Veh. Control 6% 65% 0.00 0 1% 23% 0.00 0

High Dose vs. 5/70 31/48 2/76 11/47 Untr. Control 7% 65% 0.00 0 3% 23% 0.00 0

High Dose vs. 26/50 31/48 4/50 11/47 Low Dose 52% 65% 0.14 5 8% 23% 0.03 4

Veh. Con. vs. 5/70 5/7 7 2/76 1/8 0 Untr. Control 7% 6% 0.686 3% 1% 0.887

(continued)

115 Table XXVII. Exact Statistical Tests Comparing Proportions of Animals (Not Adjusted for Age) with Observed Hepatocellular Carcinoma or Neoplastic Nodule of the Liver among Pooled Control and Trichloroethylene-Treated Mice (continued)

Comparison Chi~square df Ρ Chi-square df Ρ

Among High Dose, Low Dose and Vehicle Control 52.02 2 0.000 17.76 2 0.000

Dose-Response Trend Vehicle Control 47.85 1 0.000 16.96 1 0.000

Deviation from Trend Vehicle Control 4.16 1 0.041 0.80 1 0.367

Among High Dose, Low Dose and Untreated Control 47.31 2 0.000 14.41 2 0.001

Dose-Response Trend Untreated Control 43.43 1 0.000 13.41 1 0.000

Deviation from Trend Untreated Control 3.89 1 0.049 0.99 1 0.344 r = Number of animals with observed mark. η = Number of animals examined histopathologically for the mark. Ρ = Exact probability of exceeding or equaling observed test statistic under null hypothesis, df = Degrees of freedom. See section on statistical methodology for explanation of tests.

116 APPENDIX D : PATHOLOGY

117 Table XXVIIIa. Numbers of Tissues Examined - Rats

Males Females Controls Low High Low High Organ Males Females Dose Dose Dose Dose

Brain 20 20 50 49 48 49 Spinal Cord Pituitary 20 20 47 47 47 49 Thyroid 20 20 50 48 46 50 Adrenal 20 20 50 49 47 49 Heart 20 20 50 50 48 50 Lung 20 20 50 50 48 50 Spleen 20 20 49 48 47 50 Liver 20 20 50 50 48 50 Kidney 20 20 50 50 48 50 Stomach 20 20 49 49 47 50 Small Intestine 18 19 49 47 46 50 Large Intestine 19 20 50 50 46 50 Pancreas 20 18 50 48 48 49 Ovary/Testes 20 20 49 47 48 49 Uterus/Prostate 16 20 37 27 48 49 Vagina/Seminal Vesicle 2 4 1 1 Salivary Gland 18 16 33 14 25 24 Lymph Node 20 20 49 39 41 44 Urinary Bladder 20 18 46 46 42 43 Gallbladder Nerve Muscle Eye 1 1 Bone 20 20 50 50 48 48 Mammary Gland 20 20 50 46 45 48 Esophagus 19 19 49 50 47 48 Trachea 20 20 50 48 48 50 Thymus-Cervical Lymph Node 17 15 31 15 22 16 Unusual Lesion 2 1 Tissue Mass 6 4 5 7 7 9 Aorta 1

Total Animals Examined 20 20 50 50 48 50

118 Table XXVIIIb. Numbers of Tissues Examined - Mice

Males Females Controls Low High Low High Organ Males Females Dose Dose Dose Dose

Brain 20 20 50 48 50 47 Spinal Cord Pituitary 16 16 35 34 44 43 Thyroid 18 20 50 47 46 45 Adrenal 20 20 50 47 49 47 Heart 20 20 50 48 49 47 Lung 20 20 50 48 50 47 Spleen 20 20 50 48 49 47 Liver 20 20 50 48 50 47 Kidney 20 20 50 48 50 47 Stomach 20 20 49 48 49 46 Small Intestine 19 20 50 48 48 47 Large Intestine 20 20 48 47 49 47 Pancreas 20 20 49 47 49 47 Ovary/Testes 20 20 50 48 47 47 Uterus/Prostate 18 20 47 47 48 47 Vagina/Seminal Vesicle 3 Salivary Gland 15 18 49 39 47 47 Lymph Node 20 19 47 45 49 47 Urinary Bladder 20 19 48 47 48 44 Gallbladder 16 16 36 15 39 34 Nerve Muscle Eye 2 1 2 Bone 20 20 50 48 49 47 Mammary Gland 20 20 50 48 49 47 Thymus 19 20 43 42 46 43 Trachea 19 20 49 48 49 46 Esophagus 18 20 50 48 49 47 Unusual Lesion 3 1 5 5 4 1

Total Animals Examined 20 20 50 48 50 47

119 Tumor Summary Tables for Rats and Mice

Tables XXIXa and XXXa were designed to summarize only the number of primary tumors in each organ of each system. These tables delineate each system, each organ within each system, and the type of tumors within each organ. The counts for each of these 3 categories, that is, the numbers of animals with tumors in both system and organ and the numbers of a particular tumor within each organ, are indented in a hierarchal manner in the same manner as are the categories in the left hand column. If an animal has more than one type of tumor within a given system the total number of animals with tumors in that system may be less than the sum of the organ counts (the number of animals with tumors in a particular organ within that system). For example, 7 high dose female rats have mammary gland tumors and 1 has a tumor of the uterus/endometrium. But since the latter tumor appeared in an animal that also had a mammary gland tumor, the total number of high dose female rats with tumors of the reproductive system is 7, not 8.

In the summary, animals examined represent the number of animals started on test in a specific group less the number of animals with information missing (animal lost or autolyzed) in the group. The number of animals with tumors may be less than the number of animals with benign tumors plus the number of animals with malignant tumors since an animal may have both a benign and a malignant tumor.

Tables XXIXb and XXXb were designed to summarize the number of tumors present in each anatomic site, that is, in each organ of each system regardless of their origin. Thus, all sites of metastatic tumors which appear in more than one organ are included. These tables are organized in the same manner as are Tables XXIXa and XXXa. The counts differ only in that both system and organ counts represent number of animals with tumor in a specific system or organ irrespective of the origin of the tumor. For example, in low dose male rats a hemangiosarcoma of the subcutaneous tissue of the integumentary system is included in Table XXX but not in Table XXIX because although the tumor is present in this tissue it is not a primary tumor of the tissue and originated elsewhere. The tumor summaries for each table are identical except that Table XXX contains the additional counts of total metastatic tumors and animals with metastatic tumors.

120 Table XXIXa. Primary Tumors by Site of Origin - Trichloroethylene-Treated Rats

Organ System Male Rats Female Rats Control Low Dose High Dose Control Low Dose High Dose

INTEGUMENTARY SYSTEM 3 1 2 Skin 1 Pilomatrixoma 1 Subcutaneous Tissue 3 2 Liposarcoma 1 Fibroma 1 1 Fibrosarcoma 1 Squamous-Cell Carcinoma 1

RESPIRATORY SYSTEM 1 Lung 1 Adenosquamous Carcinoma 1

CIRCULATORY SYSTEM 1 1 2 1 Subcutaneous Tissue 1 Hemangiosarcoma 1 Multiple Organs 1 Hemangiosarcoma 1 Pancreas 1 Hemangiosarcoma 1 Spleen 1 1 Hemangiosarcoma 1 1

DIGESTIVE SYSTEM None

Continued on Next Page Table XXIXa (continued)% Primary Tumors by Site of Origin - Trichloroethylene-Treated Rats

Organ System Male Rats Female Rats Control Low Dose High Dose Control Low Dose High Dose

URINARY SYSTEM 2 2 Kidney 2 2 Malignant Mixed Tumor 2 Tubular Adenocarcinoma 1 Hamartoma 1 1

ENDOCRINE SYSTEM 1 2 1 4 3 7 Pituitary 4 2 6 Chromophobe Adenoma 4 2 6 Adrenal 1 Adrenal Cortical Carcinoma 1 Thyroid 1 2 1 1 Follicular-Cell Adenoma 1 1 Follicular-Cell Adenocarcinoma 1 1 1

HEMATOPOIETIC SYSTEM 1 1 1 Multiple Organs 1 Reticulum-Cell Sarcoma 1 Spleen 1 Reticulum-Cell Sarcoma 1 Thymus 1 Reticulum-Cell Sarcoma 1

REPRODUCTIVE SYSTEM 5 6 7 Mammary Gland 4 5 7 Fibroadenoma 3 5 7 Adenocarcinoma 1 Uterus/Endometrium 1 1 Sarcoma 1 1 Ovary 1 Granulosa-Cell Carcinoma 1

Continued on Next Page Table XXIXa (continued). Primar y Tumors by Site of Origin - Trichloroethylene-Treated Rats

Organ System Male Rats Female Rats Control Low Dose High Dose Control Low Dose High Dose

NERVOUS SYSTEM 1 Heart 1 Aortic Body Tumor 1

MUSCULOSKELETAL SYSTEM None

ALL OTHER SYSTEMS. 1 Abdomen 1 Giant-Cell Tumor, Malignant 1

PRIMARY TUMOR SUMMARY Animals Examined 20 50 50 20 48 50 Animals with Benign Tumors 2 3 2 6 11 Total Benign Tumors 2 3 2 7 8 13 Animals with Malignant Tumors 5 5 3 2 5 3 Total Malignant Tumors 5 5 3 3 6 3 Animals with Tumors 5 7 5 7 12 12 Table XXIXb. Tumors by Anatomic Site - Trichloroethylene-Treated Rats

Organ System Male Rats Female Rats Control Low Dose High Dose Control Low Dose High Dose

INTEGUMENTARY SYSTEM 4 1 2 Skin 1 Pilomatrixoma 1 Subcutaneous Tissue 4 2 Liposarcoma 1 Hemangiosarcoma 1 Fibroma 1 1 Fibrosarcoma 1 Squamous-Cell Carcinoma 1

RESPIRATORY SYSTEM 1 1 Lung 1 1 Hemangiosarcoma 1 Adenosquamous Carcinoma 1

CIRCULATORY SYSTEM 1 1 Heart 1 1 Hemangiosarcoma 1 Aortic Body Tumor 1

DIGESTIVE SYSTEM 1 1 Liver 1 Reticulum-Cell Sarcoma 1 Pancreas 1 Hemangiosarcoma 1

Continued on Next Page Table XXIXb (continued). Tumors by Anatomic Site - Trichloroethylene-Treated Rats

Organ System Male Rats Female Rats Control Low Dose High Dose Control Low Dose High Dose

DIGESTIVE SYSTEM 1 1 Liver 1 Reticulum-Cell Sarcoma 1 Pancreas 1 Hemangiosarcoma 1

URINARY SYSTEM 3 2 Kidney 3 2 Malignant Mixed Tumor 2 Tubular Adenocarcinoma 1 Hamartoma 1 1 Adenosquamous Carcinoma Metast. 1

ENDOCRINE SYSTEM 1 2 1 4 3 7 Pituitary 4 2 6 Chromophobe Adenoma 4 2 6 Adrenal 1 Adrenal Cortical Carcinoma 1 Thyroid 1 2 1 1 Follicular-Cell Adenoma 1 1 Follicular-Cell Adenocarcinoma 1 1 1

HEMATOPOIETIC SYSTEM 2 1 1 1 1 Spleen 1 1 1 Hemangiosarcoma 1 1 Reticulum-Cell Sarcoma 1 Cervical Lymph Node 1 1 Reticulum-Cell Sarcoma 1 Adenosquamous Carcinoma, Metast . 1 1 ι iiy iiiub j. Reticulum-Cell Sarcoma 1

Continued on Next Page Table XXIXb (continued) . Tumors by Anatomic Site - Trichloroethylene-Treated Rats

Organ System Male Rats Female Rats Control Low Dose High Dose Control Low Dose High Dose

REPRODUCTIVE SYSTEM 5 6 7 Mammary Gland 4 5 7 Fibroadenoma 3 5 7 Adenocarcinoma 1 Uterus/Endometrium 1 1 Sarcoma 1 1 Ovary 1 Granulosa-Cell Carcinoma 1

NERVOUS SYSTEM None

MUSCULOSKELETAL SYSTEM None

ALL OTHER SYSTEMS 1 Abdomen 1 Giant-Cell Tumor, Malignant 1

TUMOR SUMMARY Animals Examined 20 50 50 20 48 50 Animals with Benign Tumors 2 3 2 6 7 11 Total Benign Tumors 2 3 2 7 8 13 Animals with Malignant Tumors 5 5 3 2 5 3 Total Malignant Tumors 5 5 3 3 6 3 Animals with Metastatic Tumors 1 Total Metastatic Tumors 2 Animals with Tumors 5 7 5 7 12 12 Table XXXa· Primary Tumors by Site of Origin ­ Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose

INTEGUMENTARY SYSTEM 3 1 1 Skin 1 Fibrosarcoma 1 Subcutaneous Tissue 2 1 1 Fibrosarcoma 2 1 Fibroma 1

RESPIRATORY SYSTEM 5 2 1 4 7 Lung 5 2 1 4 7 Adenoma 5 1 1 2 5 Alveolar Adenocarcinoma 1 2 2

CIRCULATORY SYSTEM 1 Lung 1 Hemangiosarcoma 1

DIGESTIVE SYSTEM 1 26 31 4 11 Stomach 1 Papilloma 1 Liver 1 26 31 4 11 Hepatocellular Carcinoma 1 26 31 4 11

URINARY SYSTEM 1 Kidney 1 Adenoma 1

Continued on Next Page Table XXXa (continued). Primary Tumors by Site of Origin - Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose

ENDOCRINE SYSTEM None

HEMATOPOIETIC SYSTEM 1 4 2 1 5 6 Thymus 1 Lymphos arcoma 1 Multiple Organs 1 4 2 3 4 Reticulum-Cell Sarcoma 1 2 1 2 2 Lymphos arcoma 2 1 1 2 Spleen 1 1 Lymphosarcoma 1 1 Cervical Lymph Node 1 Malignant Lymphoma 1 Mesentery Lymph Node 1 Reticulum-Cell Sarcoma 1

REPRODUCTIVE SYSTEM 1 4 Mammary Gland 1 Adenocarcinoma 1 Uterus 1 1 Fibrosarcoma 1 Adenocarcinoma 1 Ovary 2 Granulosa-Cell Carcinoma 1 Cystadenoma 1

NERVOUS SYSTEM 1 Muscle of Back 1 Neurofibroma 1

Continued on Next Page Table XXXa (continued). Primary Tumors by Site of Origin - Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose

MUSCULOSKELETAL SYSTEM 1 Soft Tissue 1 Osteosarcoma 1

SPECIAL SENSE ORGANS 1 1 Harderian Gland 1 1 Adenoma 1 1

ALL OTHER SYSTEMS 1 Abdomen 1 Fibrosarcoma 1

PRIMARY TUMOR SUMMARY Animals Examined 20 50 48 20 50 47 Animals with Benign Tumors 7 4 1 3 5 Total Benign Tumors 7 4 1 4 5 Animals with Malignant Tumors 5 28 32 3 14 16 Total Malignant Tumors 5 30 36 3 15 19 Animals with Tumors 5 30 33 4 14 19 Table XXXb. Tumors by Anatomic Site - Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose

INTEGUMENTARY SYSTEM 3 1 1 1 1 Skin 1 1 Fibrosarcoma 1 Alveolar Adenocarcinoma, Metast. 1 Subcutaneous Tissue 2 1 1 Fibrosarcoma 2 1 Fibroma 1 Soft Tissue 1 Osteosarcoma 1

RESPIRATORY SYSTEM 9 5 1 4 8 Lung 9 5 1 4 8 Adenoma 5 1 1 2 5 Alveolar Adenocarcinoma 1 2 2 Hemangiosarcoma 1 Reticulum-Cell Sarcoma 1 Lymphosarcoma 1 Hepatocellular Carcinoma, Metast. 4 3

CIRCULATORY SYSTEM 1 Aorta 1 Alveolar Adenocarcinoma, Metast. 1

DIGESTIVE SYSTEM 2 28 32 7 14 Stomach 2 1 Papilloma 1 Reticulum-Cell Sarcoma 1 Fibrosarcoma Metastatic 1 Ileum 1 1 1 Reticulum-Cell Sarcoma 1 1 Lymphosarcoma 1

Continued on Next Page Table XXXb (continued). Tumors by Anatomic Site - Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose

REPRODUCTIVE SYSTEM 1 2 1 6 Mammary Gland 1 Adenocarcinoma 1 Uterus 1 ? Fibrosarcoma 1 Reticulum-Cell Sarcoma 1 Adenocarcinoma 1 Ovary 2 Granulosa-Cell Carcinoma 1 Cystadenoma 1 Vagina 1 Reticulum-Cell Sarcoma 1 Epididymis 1 Reticulum-Cell Sarcoma 1 Prostate 1 2 Reticulum-Cell Sarcoma 1 1 Lymphosarcoma 1 Seminal Vesicle 1 Reticulum-Cell Sarcoma 1

NERVOUS SYSTEM

None

MUSCULOSKELETAL SYSTEM 1 Muscle of Back 1 Neurofibroma 1

SPECIAL SENSE ORGANS 1 1 Harderian Gland 1 1 Adenoma 1 1

Continued on Next Page Table XXXb (continued). Tumors by Anatomic Site - Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose Hig h Dose

DIGESTIVE SYSTEM (cont.) Pancreas 2 Reticulum-Cell Sarcoma 1 Fibrosarcoma Metastatic 1 Liver 2 28 32 6 12 Hepatocellular Carcinoma 1 26 31 4 11 Reticulum-Cell Sarcoma 1 1 2 Lymphosarcoma 2 1

URINARY SYSTEM 1 4 2 Kidney 1 4 2 Adenoma 1 Reticulum-Cell Sarcoma 1 1 Lymphosarcoma 1 1 1 Fibrosarcoma Metastatic 1

ENDOCRINE SYSTEM 1 1 Adrenal 1 1 Lymphosarcoma 1 Fibrosarcoma Metastatic 1

HEMATOPOIETIC SYSTEM 1 4 4 1 4 6 Thymus 1 1 Lymphosarcoma 1 1 Spleen 1 3 2 3 4 Reticulum-Cell Sarcoma 1 1 1 1 1 Lymphosarcoma 2 1 2 3 Lymph Node 1 2 4 1 2 3 Malignant Lymphoma 1 Lymphosarcoma 2 1 2 2 Reticulum-Cell Sarcoma 1 2 1 1 1 1 Alveolar Adenocarcinoma, Metast . 1 Fibrosarcoma Metastatic 1 Bone Marrow 1 Lvmuhosarcoma 1

Continued on Next Page Table XXXb (continued). Tumors by Anatomic Site - Trichloroethylene-Treated Mice

Organ System Male Mice Female Mice Control Low Dose High Dose Control Low Dose High Dose

ALL OTHER SYSTEMS 1 Abdomen 1 Fibrosarcoma 1

TUMOR SUMMARY Animals Examined 20 50 48 20 50 47 Animals with Benign Tumors 7 4 1 3 5 Total Benign Tumors 7 4 1 4 5 Animals with Malignant Tumors 5 28 32 3 14 16 Total Malignant Tumors 5 30 36 3 15 19 Animals with Metastatic Tumors 4 4 Total Metastatic Tumors 4 11 Animals with Tumors 5 30 33 4 14 19 13 3 Tables XXXI and XXXII - Individual Pathology

Index

Page

Rats

Control males 135 Low dose males 137 High dose males 142 Control females 147 Low dose females 149 High dose females 153

Mice

Control males 158 Low dose males 160 High dose males 164 Control females 168 Low dose females 169 High dose females 173

Disposition Cod e

NATD Natural Death

Τ SAC Terminal Sacrifice

MISS Missing

MSAC Moribund Sacrifice

ACCK Accident

134 Table XXXIa. Individual Pathology - Trichloroethylene-Treated Male Rats

Control Group (Vehicle) WEEKS ON DISP ANIMAL TUMORS OTHER PATH0L03Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

67 NATD 003 LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS PATTY 70 NATD 011 SPLEEN HEMANGIOSARCOMA PITUITARY INFLAMMATIO N BRONCHUS BRONCHIECTASIS 76 NATD 014 SPLEEN HEMATOPOIESIS EXTRAMED. LUNG INFLAMMATION CHRONIC THYMUS INFLAMMATION SALIVARY GLAND INFLAMMATION KIDNEY ABSCESS 76 NATD 015 LUNG INFLAMMATION CHRONIC KIDNEY INFLAMMATION CHRONIC 82 NATD 009 LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 82 NATD 013 LUNG INFLAMMATION CHRONIC 83 NATD 018 BRONCHUS BRONCHIECTASIS STOMACH CALCIUM DEPOSITION MESENTERIC LYMPH NOD E POLYARTERITIS NODOSA PANCREAS POLYARTERITIS NODOSA TESTIS ATROPHY 87 NATD 016 LUNG INFLAMMATION CHRONIC TESTIS ATROPHY 88 NATD 005 LUNG INFLAMMATION CHRONIC 88 NATD 012 LUNG INFLAMMATION CHRONIC KIDNEY INFLAMMATION CHRONIC 90 NATD 002 ABDOMEN GIANT-CELL TUMOR, MALIGNANT LUNG INFLAMMATION CHRONIC TESTIS ATROPHY, EILATERAL 91 NATD 004 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY INFLAMMATION CHRONIC PROSTATE INFLAMMATION SEMINAL VESICLE INFLAMMATION SUBCUT TISSUE ABSCESS

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

Control Group (Vehicle)

WEEKS ON DIS P ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGBAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

96 NAT D 019 KIDNE Y MALIGNANT MIXED TUMOR THYROID FOLLICULAR ADENOMA LUNG INFLAMMATION CHRONIC 98 NAT D 020 EPIDIDYMIS FAT NECROSIS WITH ENCAPSULATION KIDNEY INFLAMMATION CHRONIC 99 NAT D 010 KIDNE Y MALIGNANT MIXED TUMOR SPLEEN HEMATOPOIESIS EXTRAMED. KIDNEY HAMARTOMA LUNG INFLAMMATION CHRONIC 102 NAT D 017 LUNG INFLAMMATION CHRONIC KIDNEY INFLAMMATION CHRONIC MESENTERIC LYMPH NOD E POLYARTERITIS NODOSA LIVER METAMORPHOSIS FATTY 103 NAT D 008 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION BONE MARROW METAMORPHOSIS FATTY 110 TSA C C01 LUNG INFLAMMATION CHRONIC KIDNEY INFLAMMATION CHRONIC SKIN ACANTHOSIS SKIN HYPERKERATOSIS SKIN INFLAMMATION 110 TSA C 006 LUNG INFLAMMATION CHRONIC KIDNEY INFLAMMATION CHRONIC THYROID INFLAMMATION CYSTIC BONE MARROW METAMORPHOSIS FATTY 110 NAT D 007 LUN G CARCINOMA, GLANDULAR AND STOMACH ULCER FOCAL SQUAMOUS, PROBABLY PRIMARY BONE MARROW METAMORPHOSIS FATTY IN LUNG, WITH MULTIPLE PULMONARY METASTASES KIDNEYS, BILATERAL MULTIPL E METASTATIC TUMORS CERVICAL LYMPH NODE METASTASI C TUMORS

end of male rat control Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOG Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

16 NATD 028 NO SIGNIFICANT DIAGNOSIS 17 NATD 036 NO SIGNIFICANT DIAGNOSIS 23 NATD 010 NO SIGNIFICANT DIAGNOSIS 27 NATD 006 NO SIGNIFICANT DIAGNOSIS 34 NATD 035 KIDNEY NEPHROSIS IOXIC BONE MARROW METAMORPHOSIS FATTY 40 NATD 030 KIDNEY NEPHROSIS IOXIC 42 NATD 034 KIDNEY NEPHROSIS IOXIC LUNG INFLAMMATION CHRONIC 48 NATD 003 KIDNEY NEPHROSIS IOXIC BONE MARROW METAMORPHOSIS FATTY 50 NATD 007 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 53 NATD 041 KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY 60 NATD 046 KIDNEY NEPHROSIS 1OXIC PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION 61 NATD 039 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION 65 NATD 019 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 67 NATD 032 KIDNEY NEPHROSIS IOXIC BRONCHUS BRONCHIECTASIS BONE MARROW METAMORPHOSIS FATTY

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOG Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

67 NATD KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 72 NATD 040 LEFT ADRENAL INFLAMMATION CYSTIC KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 74 NATD 047 KIDNEY NEPHROSIS IOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 76 NATD 018 LIVER INFLAMMATION CYSTIC KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 00 76 NATD 023 THYROID FOLLICULAR ADENOMA PARATHYROID HYPERPLASIA AORTA ARTERIOSCLEROSIS KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC STOMACH CALCIUM DEPOSITION PANCREAS POLYARTERITIS NODOSA BONE MARROW METAMORPHOSIS FATTY 80 NATD 026 SUBCUT TISSUE/NECK HEMANGIOSARCOMA ADRENAL AN3IECTASIS KIDNEY NEPHROSIS TOXIC KIDNEY CAPSULAR ABSCESS LUNG INFLAMMATICN CHRONIC STOMACH ULCER FOCAL 80 NATD 033 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC MESENTERY POLYARTERITIS NODOSA PANCREAS POLYARTERITIS NODOSA 82 NATD 005 KIDNEY NEPHROSIS IOXIC LUNG INFLAMMATION CHRONIC 83 NATD 038 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TESTIS ATROPHY BONE MARROW METAMORPHOSIS FATTY 83 NATD 043 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATICN CHRONIC

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOG Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

85 NATD 001 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TRACHEA INFLAMMATION

86 NATD 014 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION TESTIS ATROPHY TRACHEA INFLAMMATION

86 NATD 020 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY

87 NATD 029 MYOCARDIUM DEGENERATION ATRIUM CALCIUM DEPOSITION AORTA ARTERIOSCLEROSIS KIDNEY NEPHROSIS IOXIC LUNG INFLAMMATION CHRONIC STOMACH CALCIUM DEPOSITION TESTIS ATROPHY VENTRICLE CALCIUM DEPOSITION

88 NATD 012 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY SKIN ACANTHOSIS SKIN HYPERKERATOSIS SKIN EPIDERMAL INCLUSION CYST

90 NATD 015. KIDNEY TUBULAR ADENOCARCINOMA, SPLEEN HEMATOPOIESIS £XTRAMED. UNILATERAL LIVER METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC MESENTERY POLYARTERITIS NODOSA PANCREAS POLYARTERITIS NODOSA RIGHT ADRENAL CONa£NITAI MALFORMATION

94 NATD 025 SUBCUT TISSUE/AXILLA FIBROMA MYOCARDIUM DEGENERATICN MYOCARDIUM FIBROSIS LIVER INFLAMMATION KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHEE PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

94 NATD 049 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC SUBCUTANEOUS TISSUE ABSCESS OF HIND LEG EPIDIDYMIS FAT NECROSIS WITH ENCAPSULATION

96 NATD 021 KIDNEY NEPHROSIS 1OXIC LUNG INFLAMMATION CHRONIC TESTIS ATROPHY

97 NATD 016 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PANCREAS POLYARTERITIS NODOSA BONE MARROW METAMORPHCSIS FATTY

102 NATD 009 KIDNEY NEPHROSIS 1CXIC LUNG INFLAMMATION CHRONIC

103 NATD 013 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

103 NATD 027 ADRENAL ANGIECTASIS ADRENAL DEGENERATION KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION TESTIS ATROPHY 103 NATD 031 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY

104 NATD 037 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PANCREAS POLYARTERITIS NODOSA TESTIS ATROPHY PROSTATE INFLAMMATION

107 NATD 008 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TRACHEA INFLAMMATION CERVICAL LYMPH NODE INFLAMMATION

107 NATD 045 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PANCREAS POLYARTERITIS NODOSA PROSTATE INFLAMMATION

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

108 NATD 004 SUBCUT TISSUE/AXILLA FIBROSARCOMA KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TESTIS ATROPHY TESTIS CALCIUM DEPOSITION 110 TSAC 002 THYROID FOLLICULAR ADENOCARCINOMA LIVER METAMORPHCSIS FATTY KIDNEY HAMARTOMA, MEDULLA, UNILATERAL LIVER INFLAMMATION CYSTIC KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TESTIS ATROPHY 110 TSAC 011 SUBCUT TISSUE/AXILLA SQUAMOUS CELL CARCINOMA KIDNEY NEPHROSIS 1OXIC LUNG INFLAMMATION CHRONIC PROSTATE INFLAMMATION ; no TSAC 017 SPLEEN HEMATOPOIESIS EXTRAMED. KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PANCREAS POLYARTERITIS NODOSA MESENTERY POLYARTERITIS NODOSA TESTIS ATROPHY 110 TSAC 022 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TESTIS ATROPHY

110 TSAC 024 LIVER METAMCRPHCSIS FATTY KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PANCREAS POLYARTERITIS NODOSA TESTIS ATROPHY BONE MARROW METAMORPHOSIS FATTY 110 NATD 042 KIDNEY NEPHROSIS TOXIC TESTIS ATROPHY 110 TSAC 048 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC TESTIS ATROPHY BONE MARROW METAMORPHOSIS FATTY 110 TSAC 050 MYOCARDIUM DEGENERATION KIDNEY NEPHROSIS TOXIC TESTIS ATROPHY BONE MARROW METAMORPHOSIS FATTY

end of male rats—low dose Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

High Dose Group

WEEKS ON DIS P ANIMA L TUMORS OTHER PATHOLOGY STUDY CODE NUMBE R TOPOGRAEHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

2 NATD 01 2 PERICARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION

2 NATD 03 2 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION BONE MARROW METAMORPHOSIS FATTY

5 NATD 01 4 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC

6 NATD 00 4 KIDNEY NEPHROSIS TCXIC

12 NAT D 01 5 LIVER/CENTRILOBULAR METAMORPHOSIS FATTY LIVER CONGESTION KIDNEY NEPHROSIS TOXIC

17 NAT D 02 2 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

21 NAT D t>0 6 KIDNEY NEPHROSIS TCXIC

21 NATD 00 2 BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC

31 NAT D 02 5 PERICARDIUM INFLAMMATION BONE MARROW METAMORPHOSIS FATTY MYOCARDIUM INFLAMMATION KIDNEY NEPHROSIS TOXIC

33 NAT. D 02 4 KIDNEY NEPHROSIS TOXIC

35 NAT D 04 0 LIVER/CENTRILOBULAR METAMORPHOSIS FATTY LIVER ANGIECTASIS

40 NAT D 03 3 KIDNEY NEPHROSIS TCXIC

42 NAT D 03 9 LIVER HEPATOCYTCKEGALY, FOCAL LIVER ANGIECTASIS BONE MARROW METAMORPHOSIS FATTY

44 NAT D 00 3 KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats

High Dose Group

WEEKS OK OIS P ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAEHY MORPHOLOGY TOPOGRAPHY" MORPHOLOGY

48 NATD 018 BONE MARROW METAMORPHOSIS PATTY KIDNEY NEPHROSIS TOXIC

49 NATO 030 LIVER METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC

52 NATD 005 LIVER ΜΕΤΑMORPHCSIS FATTY BRONCHUS BRONCHIECTASIS KIDNEY NEPHROSIS TOXIC

52 NATD 041 LIVER/CENTRILOBULAR DEGENERATION LIVER METAMORPHOSIS FATTY LIVER ANGIECTASIS BRONCHUS BRONCHIECTASIS BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TCXIC EPIDIDYMIS FAT NECROSIS WITH ENCAPSULATION

53 NATD 037 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC EPIDIDYMIS FAT NECROSIS WITH ENCAPSULATION

54 NATD O1'3 BRONCHUS BRONCHIECTASIS LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

54 NATD 017 LIVER ANGIECTASIS KIDNEY NEPHROSIS TOXIC

56 NATD 027 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION THYROID INFLAMMATION KIDNEY NEPHROSIS ΤΟΠΟ

58 NATD 047 LIVER/CENTRILOBULAR DEGENERATIO N LIVER METAMORPHOSIS FATTY BRONCHUS BRONCHIECTASIS KIDNEY NEPHROSIS TOXIC

59 NATD 009 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

61 NATD 029 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats High Dose Group

WEEKS ON DIS P ANIMA L TUMORS OTHER PATHOLOGY STUDY COD E NUMBE R TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

62 NAT D 00 7 LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC 62 NAT D 03 8 ADRENAL ANGIECTASIS ADRENAL DEGENERATION LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS 1OXIC LIVER INFLAMMATION, DIFFUSE 65 NAT D 01 1 THYROID FOLLICULAR ADENOCARCINOMA BRONCHUS ABSCESS BRONCHUS BRONCHIECTASIS PLEURA INFLAMMATION BONE MARROW METAMORPHOSIS FATTY LIVER AN3IECTASIS KIDNEY NEPHROSIS TOXIC 65 NAT D 02 8 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS 1OXIC BONE MARROW METAMORPHOSIS FATTY 65 NAT D 04 5 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS IOXIC TESTIS ATROPHY BONE MARROW METAMORPHOSIS FATTY 66 NAT D 04 9 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY 68 NAT D 01 0 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC 70 NAT D 02 0 KIDNEi NEPHROSIS TOXIC EPIDIDYMIS FAT NECROSIS WITH ENCAPSULATION 71 NAT D 01 6 KIDNEY NEPHROSIS 1CXIC 72 NAT D 03 4 SKIN PILOMATRIXOMA PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHEU PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

72 NATD 050 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC ABDOMEN FAT NECROSIS WITH ENCAPSULATION 75 NATD 044 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 76 NATD 021 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC 82 NATD 019 HEART AORTIC BODY TUMOR LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 83 NATD 043 PITUITARY ANGIECTASIS LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC KIDNEY INFLAMMATION BONE MARROW METAMORPHOSIS FATTY 88 NATD 031 HEART THROMBOSIS ENDOCARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC LUNG ABSCESS KIDNEY NEPHROSIS TOXIC 91 NATD 036 PANCREAS HEMANGIOSARCOMA PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC PROSTATE INFLAMMATION 97 NATD 042 ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC TESTIS ATROPHY 99 NATD 008 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIa (continued). Individual Pathology - Trichloroethylene-Treated Male Rats High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STODY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

102 NATD 035 LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC 103 NATD 001 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 109 NATD 048 ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC KIDNEY PYELONEPHRITIS 110 TSAC 023 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 110 TSAC 026 SPLEEN HEMANGIOSARCOMA LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 110 TSAC 046 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC TESTIS ATROPHY

end of male rats—high dose Table XXXIb. Individual Pathology - Trichloroethylene-Treated Female Rats

Control Group (Vehicle)

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAEHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

25 NATD 008 LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS PATTY 47 NATD 016 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION U7 NATO 020 LUNG INFLAMMATION CHRONIC SPLEEN HEMATOPOIESIS EXTRAMSD» 6 6 NATD 006 PITUITARY CHROMOPHOBE ADENOMA LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 79 NATD 001 LUNG INFLAMMATION CHRONIC LUNG ABSCESS 87 NATD 007 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION 97 NATD 011 MAMMARY GLAND FIBROADENOMA KIDNEY INFLAMMATION CHRONIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION STOMACH ULCER FOCAL 98 NATD 015 LUNG INFLAMMATION CHRONIC 99 NATD 002 LUNG INFLAMMATION CHRONIC 102 NATD 014 PITUITARY CHROMOPHOBE ADENOMA ADRENAL ANGIECTASIS MAMMARY GLAND ADENOCARCINOMA LUNG INFLAMMATION CHRONIC 104 NATD 019 PITUITARY CHROMOPHOBE ADENOMA KIDNEY INFLAMMATION CHRONIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 108 NATD 012 KIDNEY INFLAMMATION CHRONIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY 110 TSAC 003 PITUITARY CHROMOPHOBE ADENOMA LUNG INFLAMMATION CHRONIC MAMMARY GLAND FIBROADENOMA PLEURA INFLAMMATION D.ONE MARROW METAMORPHOSIS FATTY

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats

Control Group (Vehicle)

WEEKS ON DISP ANIMAL TUHORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

110 TSAC 004 SPLEEN RETICULUM CELL SARCOMA LUNG INFLAMMATION CHRONIC OVARY GRANULOSA CELL CARCINOMA 110 TSAC 005 LUNG INFLAMMATION CHRONIC 110 TSAC 009 LUNG INFLAMMATICN CHRONIC 110 TSAC 010 MAMMARY GLAND FIBROADENOMA L£FT ADRENAL ANGIECTASIS ADRENAL DEGENERATION LUNG INFLAMMATION CHRONIC 110 TSAC 013 KIDNEY INFLAMMATICN CHRONIC LUNG INFLAMMATION CHRONIC 110 TSAC 017 LUNG INFLAMMATICN CHRONIC BONE MARROW METAMORPHOSIS FATTY 110 TSAC 018 KIDNEY INFLAMMATION CHRONIC KIDNEY INFLAMMATION CYSTIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION LIVER METAMORPHOSIS FATTY LIVER INFLAMMATION PANCREAS ATROPHY PANCREAS INFLAMMATION CYSTIC

end of female rats controls Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats Low Dose Group

WEEKS ON DIS P ANIMA L TUMORS OTHER PATHOLOG Y STUDY COD E NUMBE R TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

2 NATD 02 4 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 2 MISS 02 9 ANIMAL MISSING 2 NATD 04 2 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 3 NATD 00 2 LUNG HEMANGIOSARCOMA, KIDNEY PYELONEPHRITIS HEART HEMANGIOSARCOMA, 5 NATD 02 0 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION 7 NATD 04 7 KIDNEY NEPHROSIS TOXIC 15 NAT D C0 1 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION 16 NAT D 03 0 KIDNEY NEPHROSIS TOXIC 16 NAT D 03 8 KIDNEY CALCIUM DEPOSITION BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC 19 NAT D 01 1 SUBCUT TISSUE ABSCESS 21 NAT D 01 2 BONE MARROW METAMORPHOSIS FATTY 21 NAT D 01 4 NO SIGNIFICANT DIAGNOSIS 21 MIS S 02 1 ANIMAL MISSING 22 NAT D 00 5 BONE MARROW METAMORPHOSIS FATTY 22 NAT D 03 9 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 25 NAT D 01 7 BONE MARROW METAMORPHOSIS FATTY 26 NAT D 03 6 KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats Low Dose Group

WEEKS ON DIS P ANIMA L TUMORS OTHER PATHOLOG Y STUDY CODE NUMBE R TOPOGRAPHY MORFHOLOGY TOPOGRAPHY MORPHOLOGY

23 NATD 03 5 KIDNEY NEPHROSIS ICXIC

33 NATD 03 3 KIDNEY NEPHROSIS TCXIC

34 NATD 01 9 KIDNEY NEPHROSIS TCXIC BONE MARROW M£TAMORPHOSI3 FATTY

40 NATD 04 5 ADRENAL ANGIECTASIS ADRENAL DEGENERATION KIDNEY NEPHROSIS TOXIC

42 NATD 02 2 LUNG INFLAMMATION CHRONIC

57 NATD 00 6 KIDNEY NEPHROSIS TOXIC

60 NATD 01 0 KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY

61 NATD 04 4 KIDNEY NEPHROSIS TOXIC BRONCHUS ABSCESS Ο PLEURA INFLAMMATION 68 NATD 02 7 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

69 NATD 02 5 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

75 NATD 01 5 LUNG INFLAMMATION CHRONIC

75 NATD 01 6 LIVE R RETICULUM CELL SARCOMA KIDNEY NEPHROSIS TOXIC CERVICAL LYMPH NODE RETICULUM CELL SARCOMA LUNG INFLAMMATION CHRONIC

77 NATD 04 8 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

86 NATD 03 4 KIDNEY NEPHROSIS TCXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION

96 NATD 02 3 ADRENA L ADRENAL CORTICAL CARCINOMA KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

96 NATD 04 6 KIDNEY NEPHROSIS ICXIC LUNG INFLAMMATION CHRONIC

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHfiit PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

98 NATD 032 SUBCUT TISSUE/CHEST LIPOSARCOM A KIDNEY NEPHROSIS 1CXIC LUNG INFLAMMATION CHRONIC 100 NATD 013 SUBCOT TISSUE/NECK FIBROMA KIDNEY NcPHRCSIS TOXIC LUNG INFLAMMATION CHRONIC 102 NATD 049 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

104 NATD 040 MAMMARY GLAND FIBROADENOMA SPLEEN HJSMATCPOIISIS EXTRAMED. KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 110 TSAC 003 TRACHEA INFLAMMATION TRACHEAL LYMPH NODE INFLAMMATION KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 110 TSAC 004 KIDNEY NEPHROSIS TCXIC LUNG INFLAMMATION CHRONIC RIGHT OVARY CYST BONE MARROW METAMORPHOSIS FATTY 1 10 TSAC 007 PITUITARY CHROMOPHOBE ADENOMA ADRENAL ANGl£iCTASIS MAMMARY GLAND FIBROADENOMA MYOCARDIUM FIBROSIS KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 110 TSAC 008 KIDNEY NEPHROSIS TCXIC LUNG INFLAMMATION CHRONIC 110 TSAC 009 ADRENAL ANGIECTASIS KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC RIGHT OVARY CYST 110 TSAC 018 MAMMARY GLAND FIBROADENOMA RIGHT ADRENAL ANGIECTASIS KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAEHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

110 TSAC 026 ENDOMETRIUM SARCOMA LEFT ADRENAL AN3IECTASIS ADRENAL DEGENERATION LEFT ADRENAL INFLAMMATION CYSTIC KIDNEY NEPHROSIS TCXIC LUNG INFLAMMATION CHRONIC OVARY CYST

110 TSAC 028 ADRENAL ANGIECTASIS ADRENAL DEGENERATION BILE DUCT INFLAMMATION PROLIFERATIVE KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC OVARY CYST

110 T3AC 031 PITUITARY CHROMOPHOBE ADENOMA KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC ENDOMETRIUM INFLAMMATION LEFT OVARY CYST RIGHT EYE CATARACT RETINA DETACHMENT

110 TSAC 037 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY

110 TSAC 041 PERICARDIUM INFLAMMATION KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION

110 TSAC 043 MAMMARY GLAND FIBROADENOMA KIDNEY NEPHROSIS TCXIC LUNG INFLAMMATION CHRONIC

110 NATD 050 MAMMARY GLAND FIBROADENOMA, (MULTIPLE - 2) KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC UTERUS INFLAMMATION BILE DUCT INFLAMMATION PROLIFERATIVE

end of female rats—low dose Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOG Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

3 NATD 013 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC 5 NATD 002 PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC 9 NATD 032 LUNG INFLAMMATION CHRONIC BONE MARROW METAMORPHOSIS FATTY KIDNEY NEPHROSIS TOXIC 21 NATD 036 KIDNEY CALCIUM DEPOSITION BONE MARROW METAMORPHOSIS FATTY 24 NATD 049 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC 28 NATD 008 KIDNEY NEPHROSIS TOXIC 28 NATD 016 KIDNEY NEPHROSIS TOXIC 30 NATD 043 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 35 NATD 040 KIDNEY NEPHROSIS TOXIC 43 NATD 038 UTERUS RETENTION FLUID 46 NATD 045 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS IOXIC 50 NATD 004 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS IOXIC KIDNEY CALCIUM DEPOSITION

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats

High Dose Group

IEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOG Y STUDY CODE NUMBER TOPOGBAFHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

53 NATD 007 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC

54 Ν ATD 041 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC

58 NATD 024 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY

59 NATD 017 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY

63 NATD 031 PERICARDIUM INFLAMMATION BRONCHUS BRONCHIECTASIS KIDNEY NEPHROSIS TOXIC

64 NATD 022 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION PERICARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION KIDNEY NEPHROSIS TOXIC KIDNEY CALCIUM DEPOSITION BONE MARROW METAMORPHOSIS FATTY

67 NATD 028 KIDNEY NEPHROSIS TOXIC LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION

69 NATD 012 I fJHG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC, MARKED

69 NATD 027 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC KIDNEY CALCIUM DEPOSITION UTERUS RETENTION FLUID BONE MARROW METAMORPHOSIS FATTY

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATH0L03Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

70 NATD 018 LONG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC SHALL INTESTINE ULC2R FOCAL 70 NATD 020 LONG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC ΒΟΙΕ MARROW METAMORPHOSIS FATTY 72 NATD οία LONG INFLAMMATION CHRONIC KIDIEY NEPHROSIS TOXIC 73 NATD 001 ADREHAL ANGIECTASIS LONG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC ΒΟΙΕ MARROW METAMORPHOSIS FATTY 73 NATD 0U2 BRONCHOS ABSCESS KIDNEY NEPHROSIS TOXIC KIDNEY CALCIUM DEPOSITION 74 NATD 039 BRONCHOS BRONCHIECTASIS KIDNEY NEPHROSIS TOXIC 82 NATD 026 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY 86 NATD 04 8 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC MESENTERIC LYMPH NODE INFLAMMATION MESENTERIC LYMPH NODE INFLAMMATION CYSTIC 89 NATD 047 ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC 95 NATD 021 LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC 96 NATD 050 PITUITARY CHROMOPHOBE ADENOMA ADRENAL ANGIECTASIS LIVER METAMORPHOSIS FATTY LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIb (continued). Individual Pathology - Trichloroethylene-Treated Female Rats

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOG Y STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

101 NATD 029 MAMMARY GLAND FIBROADENOMA LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

101 NATD 035 PITUITARY CHROMOPHOBE ADENOMA ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC

102 NATD 019 LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC

103 NATD 033 MAMMARY GLAND FIBROADENOMA (MULTIPLE-3) LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS IOXIC

104 NATD 034 BRAIN HYDROCEPHALUS PERICARDIUM INFLAMMATION LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS IOXIC

110 TSAC 003 ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC BILE DUCT DILATATION KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY

110 TSAC 005 PITUITARY CHROMOPHOBE ADENOMA ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC BONE MARROW METAMORPHOSIS FATTY

110 TSAC 006 THYMUS RETICULUM CELL SARCOMA LUNG INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC OVARY INFLAMMATION

110 TSAC 009 PITUITARY CHROMOPHOBE ADENOMA ADRENAL DEGENERATION ΜΑΜΜΑΡΥ 3LAND FIBROADENOMA ADRENAL ANGIECTASIS LUNG INFLAMMATION CHRONIC PLEURA INFLAMMATION KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIb (continued) Individual Pathology - Trichloroethylene-Treated Female Rats High Dose Group

end of female rats Table XXXIIa. Individual Pathology - Trichloroethylene-Treated Male Mice Control Group (Vehicle)

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

32 NATD 010 NO SIGNIFICANT DIAGNOSIS 39 NATD 009 SPLEEN AMYLOIDOSIS KIDNEY PYELONEPHRITIS 60 NATD 008 KIDNEY INFLAMMATION CHRONIC KIDNEY HYDRONEPHROSIS KIDNEY AMYLOIDOSIS 61 NATD 007 KIDNEY INFLAMMATION CHRONIC KIDNEY AMYLOIDOSIS 64 NATD 006 KIDNEY INFLAMMATION CHRONIC KIDNEY HYDRONEPHROSIS SPLEEN AMYLOIDOSIS 66 NATD 020 MESENTERIC LYMPH NOD E ANGIECTASIS SUBCUT TISSUE ABSCESS 68 NATD 005 KIDNEY IKFLAMMATICN CHRONIC KIDNEY AMYLOIDOSIS KIDNEY HYDRONEPHROSIS SPLEEN AMYLOIDOSIS LIVER HYPERPLASIA ENDOCARDIUM HYPERPLASIA 72 NATD 019 LIVER HEPATOCELLULAR CARCINOMA SPLEEN AMYLOIDOSIS 76 NATD 004 KIDNEY INFLAMMATION CHRONIC KIDNEY HYDRONEPHROSIS 76 «SAC 018 LIVER RETICULUM CELL SARCOMA SPLEEN RETICULUM CELL SARCOMA MESENTERIC LYMPHNODE RETICULU M CELL SARCOMA PROSTATf RETICULUM CELL SARCOMA SEMINAL VESICLES RETICULUM CELL SARCOMA 77 NATD 017 SUBCOT TISSUE/BACK FIBROSARCOMA SPLEEN AMYLOIDOSIS KIDNEY INFLAMMATION CHRONIC KIDNEY AMYLOIDOSIS

Continued on next page Table XXXIIa (continued). Individual Pathology - Trichloroethylene-Treated Male Mice

Control Group (Vehicle)

WEEKS ON DISP ANIMAL TOHORS OTHSR PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY HORPHOLOGY TOPOGRAPHY MORPHOLOGY

78 NATD 003 NO SIGNIFICANT DIAGNOSIS 90 TSAC 001 SKIN OP CHEST FIBROSARCOMA KIDNEY INFLAMMATION CHRONIC KIDNEY HYDRONEPHROSIS 90 TSAC 002 KIDNEY INFLAMMATICN CHRONIC KIDNEY HYDRONEPH5OSIS 90 TSAC 011 SOBCUT TISSUE/BACK FIBROSARCOMA 90 TSAC 012 NO SIGNIFICANT DIAGNOSIS 90 TSAC 013 SKIN ACANTHOSIS SKIN INFLAMMATION 90 TSAC 014 NO SIGNIFICANT DIAGNOSIS 90 TSAC 015 KIDNEY HYDRONEPHROSIS KIDNEY INFLAMMATION CHRONIC KIDNEY AMYLOIDOSIS 90 TSAC 016 NO SIGNIFICANT DIAGNOSIS

end of male mice controls Table XXXIIa (continued). Individual Pathology - Trichloroethylene-Treated Male Mice

Low Dose Group

WEEKS ON DISP ANIMA L TUMORS OTHER PATHOLOGY STUDY CODE NUMBE R TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

16 NATD 010 NO SIGNIFICANT DIAGNOSIS 18 KATD 050 THYMUS LYMPHOSARCOMA KIDNEY NEPHROSIS TOXIC BRONCHIAL LYMPHNODE LYMPHOSARCOMA LUNG LYMPHOSARCOMA KIDNEY LYMPHOSARCOMA PROSTATE LYMPHOSARCOMA BONE MARROW LYMPHOSARCOMA CERVICAL LYMPHNODE LYMPHOSARCOMA SPLEEN LYMPHOSARCOMA LIVER LYMPHOSARCOMA 31 ACCK 009 NO SIGNIFICANT DIAGNOSIS 51 NATD 030 KIDNEY NEPHROSIS TOXIC 53 NATD 040 KIDNEY NEPHROSIS 1OXIC 58 NATD 008 HEART ORGANIZED THHCMBUS ENDOCARDIUM INFLAMMATION MYOCARDIUM INFLAMMATION KIDNEY PYELONEPHRITIS KIDNEY HYDRONEPHROSIS SPLEEN AMYLOIDOSIS URINARY BLADDER INFLAMMATION PROSTATE INFLAMMATION PANCREAS ATROPHY PANCREAS INFLAMMATIOH BONE INFLAMMATION KIDNEY NEPHROSIS IOXIC 63 NATD 039 KIDNEY NEPHROSIS TOXIC 65 NATD 020 LIVER HYPERPLASIA KIDNEY NEPHROSIS TOXIC 65 NATD 038 KIDNEY NEPHROSIS TOXIC 77 NATD 019 BRONCHUS ABSCESS CERVICAL LYMPH NODE INFLAMMATION KIDNEY NEPHROSIS TOXIC 81 NATD 028 SPLEEN LYMPHOSARCOMA KIDNEY NEPHROSIS TOXIC LIVER LYMPHOSARCOMA

Continued on next page Table XXXIIa (continued). Individua l Pathology - Trichloroethylene-Treated Male Mice Low Dose Group

WEEKS ON DISP ANIBAL TUHORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

81 NATD 029 LONG ADENOMA KIDNEY NEPHROSIS TOXIC LIVER HEPATOCELLULAR CARCINOMA

66 NATD 049 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS ICXIC STOMACH HYPERKERATOSIS STOMACH ACANTHOSIS

88 NATD 007 LIVE R HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC LUNG HEPATOCELLULAR CARCINOMA METASTA LONG ADENOMA

88 NATD 018 MESENTERIC LYMPH NODE ANGIECTASIS KIDNEY NEPHROSIS TOXIC

90 TSAC 001 LIVE R HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS ICXI C LONG ADENOMA 90 TSAC 002 S0BC0 T TISSUE/BACK FIBEOM A KIDNEY NEPHROSIS TCXIC

90 TSAC 003 LIVE R HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC

90 TSAC 004 LIVE R HEPATOCELLULAR CARCINOMA SPLEEN AMYLOIDOSIS LONG ADENOMA KIDNEY NEPHROSIS TOXIC HESENTERIC LYHPHNODE RETICULU M CELL SARCOMA ILEUM RETICULUM CELL SARCOMA RENAL LYHPHNODE RETICULUM CELL SARCOMA

90 TSAC 005 LIVE R HEPATOCELLULAR CARCINOMA LIVER THROMBOSIS SPLEEN HEMATOPOIESIS EXIRAMED. KIDNEY NEPHROSIS TOXIC

90 TSAC 006 LIVE R HEPATOCELLULAR CARCINOMA MESENTERIC LYMPH NODi: ANGIECTASIS KIDNEY INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 90 TSAC 011 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC LUNG HEPATOCELLULAR CARCINOMA METASTA 90 TSAC 012 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC 90 TSAC 013 LIVER HEPATOCELLULAR CARCINOMA MESENTERIC LYMPH NODE INFLAMMATION KIDNEY NEPHROSIS TOXIC 90 TSAC 014 HARDERIA N GLAND ADENOMA KIDNEY NEPHROSIS ICXIC

Continued on next, page Table XXXIIa (continued) . Individual Pathology - Trichloroethylene-Treated Male Mice

Low Dose Group

WEEKS ON DIS P ANIMA L TUMORS OTHER PATHOLOG Y STUDY CODE NUMBE R TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

90 TSAC 01 5 EPIDIDYMIS RETICULUM CELL SAHCOHA KIDNEY NEPHROSIS TOXIC PROSTATE RETICULUM CELL SARCOHA SPLEEN RETICULUM CELL SAfiCOHA LIVER HEPATOCELLULAR CABCIIOHA LUNG HEPATOCELLULAR CARCI1OHA BETASΓA

90 TSAC 01 6 KIDNEY NEPHROSIS TOXIC 90 TSAC 01 7 LIVER HEPATOCELLULAR CAECIBOHA KIDNEY NEPHROSIS TOXIC 90 TSAC 02 1 LIVER HEPATOCELLULAR CA ECHO HA KIDNEY NEPHROSIS TOXIC 90 TSAC 02 2 KIDNEY NEPHROSIS TOXIC 90 TSAC 02 3 KIDNEY NEPHROSIS TOXIC 90 TSAC 02 4 LIVER HEPATOCELLULAR C ARCI HO HA KIDNEY NEPHROSIS TOXIC 90 TSAC 02 5 KIDNEY NEPHROSIS TOXIC 90 TSAC 02 6 LIVER HEPATOCELLULAR CABCIIOHA KIDNEY NEPHROSIS TOXIC 90 TSAC 02 7 KIDNEY NEPHROSIS TOXIC 90 TSAC 03 1 LIVER HEPATOCELLULAR CA EC 110 HA KIDNEY NEPHROSIS TOXIC 90 TSAC 03 2 LIVER HEPATOCELLULAR CABCIIOHA KIDNEY NEPHROSIS TOXIC 90 TSAC 03 3 LIVER HEPATOCELLULAR CAECIIOHA KIDNEY NEPHROSIS TOXIC 90 TSAC 03 4 LIVER HEPATOCELLULAR CABCIIOHA KIDNEY NEPHROSIS TOXIC 90 TSAC 03 5 LIVER HEPATOCELLULAR CAHCIBGHA KIDNEY INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 90 TSAC 03 6 LIVER HEPATOCELLULAR CABCI1CHA KIDNEY NEPHROSIS TOXIC 90 TSAC 03 7 LUNG ADENOMA KIDNEY NEPHROSIS TOXIC LIVER HEPATOCELLULAR CABCIIOHA

Continued on next page Table XXXIIa (continued). Individual Pathology - Trichloroethylene-Treated Male Mice

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

90 TSAC 041 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC STOMACH ACANTHOSIS STOMACH HYPERKERATOSIS MESENTERIC LYMPH NODE INFLAMMATION RIGHT EYE CONGENITAL MALFORMATION RIGHT EYE PHTHISIS EULEI HARDERIAN GLAND INFLAMMATION 90 TSAC 042 KIDNEY NEPHROSIS TOXIC 90 TSAC 043 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC LUNG HEPATOCELLULAR CARCINOMA METASTA STOMACH ACANTHOSIS STOMACH HYPERKERATOSIS STOMACH INFLAMMATION EXUDATIVE MESENTERIC LYMPH NODE INFLAMMATION 90 TSAC 044 KIDNEY NEPHROSIS TOXIC 90 TSAC 045 KIDNEY NEPHROSIS TOXIC LIVER HYPERPLASIA 90 TSAC 046 KIDNEY NEPHROSIS TOXIC 90 TSAC 047 KIDNEY NEPHROSIS TCXIC LIVER INFLAMMATION 90 TSAC 048 LIVEB HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

end of male mice—low dose {continued)^ Individual Pathology - Trichloroethylene-Treated Male Mice

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

13 NATD 010 KIDNEY NEPHROSIS TOXIC

14 NATD 040 KIDNEY NEPHROSIS TOXIC

15 NATD 049 KIDNEY NEPHROSIS ICXIC

15 ACCK 050 NO SIGNIFICANT DIAGNOSIS

24 NATD 019 KIDNEY NEPHROSIS TOXIC

24 NATD 020 LIVER HYPfiRPLASIA KIDNEY NEPHROSIS TOXIC

25 NATD 047 KIDNEY NEPHROSIS TOXIC

25 NATD 048 KIDNEY NEPHRCSIS TOXIC

27 NATD 046 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

28 NATD 018 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NiiPHROSIS TOXIC

28 NATD 045 KIDNEY N£PHROSIS ICXIC

29 NATD 030 KIDNEY INFLAMMATION CHRONIC

30 NATD 009 KIDNEY NEPHRCSIS TCXIC

30 NATD 017 LIVER HEPATOCELLULAR CABCINCMA KIDNEY INFLAMMATION

36 NATD 008 LIVER HEFATOCELLULAR CARCINOMA KIDNEY NEPHRCSIS TCXIC

42 NATD 007 KIDNEY NEPHRCSIS TOXIC

53 NATD 039 LIVER HEPATOCELLULAR CAFCINCMA KIDNEY NEPHROSIS TOXIC

60 NATD 005 KIDNEY NEPHROSIS TCXIC

60 NATD 006 KIDNEY NEPHROSIS TOXIC

61 . NATD 016 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NSPHROSIS TOXIC 70 NATD 038 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC

71 NATD 015 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIIa (continued). Individual Pathology - Trichloroethylene-Treated Male Mice

High Dose Group _____^_

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

72 NATD 037 AUTOLYSIS 74 NATD 029 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC LIVER/CENTRILOBULAR NECROSIS 75 NATD 004 KIDNEY NEPHROSIS TOXIC LIVER/CENTRILOBULAR NECROSIS SALIVARY GLAND INFLAMMATION CYSTIC STOMACH ACANTHOSIS STOMACH HYPERKERATOSIS 75 NATD 036 AUTOLYSIS 78 MSAC 044 IIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC TISSUE MUSCLE OF NEUROFIBROMA BACK 83 NATD 035 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC LUNG HEPATOCELLULAR CARCINOMA METASTA LUNG ALVEOLAR ADENOCARCINOMA BRONCHIAL LYMPH NODE CARCINOMA METASTATIC SKIN OF CHEST CARCINOMA METASTATIC AORTA CARCINOMA METASTATIC 88 MSAC 003 LIVER HEPATOCELLULAR CARCINOMA TESTIS ATROPHY SPLEEN LYMPHOSARCOMA HARDERIAN GLAND INFLAMMATION MESENTERIC LYMPHNODE LYMPHOSARCOMA KIDNEY NEPHROSIS TOXIC: KIDNEY LYMPHOSARCOMA 90 TSAC 001 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 90 Τ SAC 002 LIVER HEPATOCELLULAR CARCINOMA THYROID HYPERPLASIA CYSTIC KIDNEY INFLAMMATION CHRONIC KIDNEY HYDRONEPHROSIS MESENTERIC LYMPH NODS INFLAMMATION KIDNEY NEPHROSIS TOXIC 90 TSAC 011 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 90 TSAC 012 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TCXIC 90 TSAd 013 KIDNEY ADENOMA LIVER HYPERPLASIA LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIIa (continued). Individual Pathology - Trichloroethylene-Treated Male Mice

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

90 TSAC 014 LUNG HEPATOCELLULAR CARCINOMA METASTA KIDNEY HYDRONEPHfCSIS LIVER HEPATOCELLULAR CARCINOMA KIDNEY INFLAMMATION CHRONIC KIDNEY NEPHROSIS TOXIC 90 TSAC 021 LIVER HEPATOCELLULAR CARCINOMA KIDNEY HYDRONEPHROSIS KIDNEY NEPHROSIS TOXIC 90 TSAC 022 LIVER HEPATOCELLULAR CARCINOMA MESENTERIC LYMPH NODE INFLAMMATION KIDNEY NEPHROSIS TOXIC 90 TSAC 023 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 9C TSAC 024 MESENTERIC LYMPHNODE RETICULUM CELL SARCOMA KIDNEY NEPHROSIS TOXIC SPLEEN RETICULUM CELL SARCOMA PANCREAS RETICULUM CELL SARCOMA KIDNEY RETICULUM CELL SARCOMA LIVER RETICULUM CELL SARCOMA 90 TSAC 025 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 90 TSAC 026 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 90 TSAC 027 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 90 TSAC 028 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 90 TSAC 031 LUNG ADENOMA KIDNEY NEPHROSIS TOXIC 90 TSAC 032 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC LUNG HEPATOCELLULAR CARCINOMA METASTA MESENTERIC LYMPH NODE ANGIECTASIS 90 TSAC 033 LIVER HEPATOCELLULAR CARCINOMA STOMACH HYPERKERATOSIS STOMACH PAPILLOMA KIDNEY NEPHROSIS TOXIC 90 NATD 034 KIDNEY FIBROSARCOMA, METASTATIC KIDNEY NEPHROSIS TOXIC LIVER HEPATOCELLULAR CARCINOMA ABDOMENAL CAVITY FIBROSARCOMA, PRIMARY ADRENAL FIBROSARCOMA, METASTATIC PANCREAS FIBROSARCOMA, METASTATIC MESENTERIC LYMPHNODE FIBROSARCOMA, METASTATIC STOMACH FIBROSARCOMA, METASTATIC

Continued on next page _ Table XXXIIa (continued). Individual Pathology - Trichloroethylene-Treated Male Mice

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MOPPHOLOGY TOPOGRAPHY MORPHOLOGY

90 TSAC 041 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

90 TSAC 042 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS ICXIC

90 TSAC 043 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC LUNG HEMANGIOSARCOMA

end of male mice—high dose Table XXXIIb. Individual Pathology - Trichloroethylene-Treated Female Mice Control Group (Vehicle)

WEEKS ON DISP ANIMAL TUMORS OTH^R PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

20 ACCK 010 NO SIGNIFICANT DIAGNOSIS 37 ACCK 009 ENDOMETRIUM HYPERPLASIA CYSTIC 83 ACCK 008 SOFT TISSUES OF OSTEOSARCOMA BACK

90 TSAC 001 OVARY CYST 90 TSAC 002 LUNG INFLAMMATION CHRONIC UTERUS INFLAMMATION OVARY CYST OVARY INFLAMMATION 90 TSAC 003 ENDOMETRIUM ADENOCARCINOMA

90 TSAC 004 NO SIGNIFICANT DIAGNOSIS 90 TSAC 00 5 ENDOMETRIUM HYPERPLASIA CYSTIC OVARY CYST 90 TSAC 006 ENDOMETRIUM HYPERPLASIA CYSTIC 90 TSAC 007 LUNG ADENOMA

90 TSAC 011 OVARY CYST ENDOMETRIUM HYPERPLASIA CYSTIC 90 TSAC 012 NO SIGNIFICANT DIAGNOSIS 90 TSAC 013 ENDOMETRIUM HYPERPLASIA CYSTIC

90 TSAC 014 MESENTERIC LYMPH NODE RETICULUM CELL SARCOMA LUNG INFLAMMATION CHRONIC ENDOMETRIUM HYPERPLASIA CYSTIC 90 TSAC 015 OVARY CYST 90 TSAC 016 ENDOMETRIUM HYPERPLASIA CYSTIC 90 TSAC 017 ENDOMETRIUM HYPERPLASIA CYSTIC 90 TSAC 018 OVARY CYST ENDOMETRIUM HYPERPLASIA CYSTIC 90 TSAC 019 NO SIGNIFICANT DIAGNOSIS

90 TSAC 020 ENDOMETRIUM HYPERPLASIA CYSTIC

end of female mice controls Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

10 NATD 020 NO SIGNIFICANT DIAGNOSIS 32 ACCK 010 KIDNEY NEPHROSIS TOXIC 37 Μ SAC 019 KIDNEY NEPHROSIS TOXIC 38 NATD 018 NO SIGNIFICANT DIAGNOSIS 4 1 ACCK 009 KIDNEY NEPHROSIS TOXIC 55 NATD 030 UTERUS INFLAMMATION 63 NATD 029 NO SIGNIFICANT DIAGNOSIS 66 NATD 040 KIDNEY NEPHROSIS TOXIC 76 NATD 017 LIVER RETICULUM CELL SARCOMA KIDNEY NEPHROSIS TOXIC UTERUS RETICULUM CELL SARCOMA CERVICAL LYMPHNODE RETICULUM CELL SARCOMA

81 NATD 016 SPLEEN LYMPHOSARCOMA KIDNEY NEPHROSIS 1CXIC MESENTERIC LYMPHNODE LYMPHOSARCOMA ILEUM LYMPHOSARCOMA ADRENAL LYMPHOSARCOMA CERVICAL LYMPHNODE LYMPHOSARCOMA

90 TSAC 00 1 LIVER RETICULUM CELL SARCOMA KIDNEY NEPHROSIS 1CXIC VA3INA RETICULUM CELL SARCOMA SPLEEN RETICULUM CELL SARCOMA

90 TSAC 002 UTERUS INFLAMMATION KIDNEY NEPHROSIS TOXIC 90 TSAC 003 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC UTERUS FIBROSARCOMA

90 TSAC 004 ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC 90 TSAC 005 KIDNEY NEPHROSIS TCXIC 90 TSAC 006 ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAFHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

90 TSAC 007 LIVER HEPATOCELLULAR CARCINOMA ENDOMETRIUM HYPEEFLASIA CYSTIC HARDERIAN SLAND HYPERPLASIA KIDNEY NEPHROSIS TOXIC

90 TSAC 008 STOMACH ACANTHOSIS STOMACH HYPERKERA1OSIS KIDNEY NEPHROSIS TOXIC

90 TSAC 011 UTERUS RETSNTION FLUID KIDNEY NEPHROSIS 1OXIC

90 TSAC 012 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

90 TSAC 013 OVARY CYST UTERUS RETENTION FLUID KIDNEY NEPHROSIS TOXIC

90 TSAC 014 UTERUS POLYP UTERUS INFLAMMATION KIDNEY NEPHROSIS TOXIC

90 TSAC 015 UTERUS RETENTION FLUID KIDNEY NEPHROSIS TOXIC

90 TSAC 021 KIDNEY NEPHHOSIS TOXIC

90 TSAC 022 KIDNEY NEPHROSIS TOXIC

90 TSAC 023 ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC

90 TSAC 024 £NDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NJSPHROSIS TOXIC

90 TSAC 025 ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NfiPHROSIS TOXIC

90 TSAC 026 KIDNEY NEPHROSIS TCXIC

90 TSAC 027 LUNG ALVEOLAR ADENOCARCINOMA KIDNEY INFLAMMATICN INTERSTITIAL KIDNEY INFLAMMATION PROLIFERATIVfi PANCREAS ATROPHY KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

90 TSAC 028 ENDOMETRIUM HYPERFLASIA CYSTIC KIDNEY NEPHROSIS TOXIC

90 TSAC 031 KIDNEY N£PHfiOSIS TCXIC

90 TSAC 032 LIVER HEPATOCELLULAR CARCINOMA UTERUS RETENTION J1UIC KIDNEY NEPHROSIS TOXIC

90 TSAC 033 OVARY GRANULOSA CELL CARCINOMA UTERUS INFLAMMATION KIDNEY NEPHfiOSIS TOXIC

90 TSAC 034 ENDOMETRIUM HYPSRPLASIA CYSTIC OVARY CYST KIDNEY NEPHROSIS TOXIC

90 TSAC 035 SUBCUT TISSU/ABDOMEN PIBROSARCOMA ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC

90 TSAC 036 LUNG ADENOMA KIDNEY NEPHfiOSIS TOXIC SPLEEN LYMPHOSARCOMA

90 TSAC 037 SNDOMETRIUM HYPSRPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC

90 TSAC 038 MAMMARY GLAND ADENOCARCINOMA OVARY CYST KIDNEY NSPHROSIS TOXIC

90 TSAC 039 KIDNEY NEPHROSIS TCXIC

90 TSAC 041 KIDNEY NEPHROSIS TCXIC

90 TSAC 042 LUNG ADENOMA OVARY RETENTION ILUID THYMUS LYMPHOSAECOMA KIDNEY NEPHROSIS TOXIC

90 TSAC 043 OVARY CYSTADENOMA THYROID INFLAMMATION CYSTIC LUNG ALVEOLAE ADENOCARCINOMA KIDNEY NEPHROSIS TOXIC HARDERIAN GLAND ADENOMA

90 TSAC 044 iSNDOMETRIUM HYPERPLASIA CYSTIC THYROID INFLAMMATION CYSTIC KIDNEY NEPHROSIS TOXIC

90 TSAC 045 UTERUS RETENTION FLUID RIGHT OVARY CYST KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice

Low Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

TSAC 046 KIDNEY NEPHROSIS TCXIC 91 TSAC 047 ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC 91 TSAC 048 UTERUS RETENTION ELUIC KIDNEY NEPHROSIS TOXIC 91 TSAC 049 ENDOMETRIUM HYPERPLASIA CYSTIC KIDNEY NEPHROSIS TOXIC 91 TSAC 050 UTERUS RETENTION fLUID KIDNEY NEPHROSIS TOXIC

end of female mice—lew dose Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice

High Dose Group

WEEKS ON DIS P ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

14 MIS S 01 0 ANIMAL MISSING 22 MIS S 04 0 ANIMAL MISSING 26 MIS S 05 0 ANIMAL MISSING 32 NATD 03 0 KIDNEY NEPHROSIS 1CXIC 32 NAT D 04 9 KIDNEY NEPdRCSIS 33 NAT D 03 9 KIDNEY NEPHROSIS 1OXIC 38 NAT D 03 8 KIDNEY NEPHROSIS TOXIC 39 NAT D 02 0 KIDNEY NEPHROSIS TOXIC UO NATD 03 7 KIDNEY N£PHROSIS TOXIC 69 NAT D 00 9 SPLEE N LYMPHOSARCOMA KIDNEY NEPHROSIS 1OXIC OVARY CYST 88 NAT D 03 6 SPLEE N RETICULUM CELL SARCOMA KIDNEY RETICULUM CELL SARCOMA LUNG RETICULUM CELL SARCOMA STOMACH RETICULUM CELL SARCOMA 91 TSA C 00 1 MESENTERI C LYMPH NODE RETICULUM CELL SARCOMA KIDNEY NEPHROSIS TOXIC ILEUM RETICULUM CELL SARCOMA 91 TSA C 00 2 KIDNEY NEPHROSIS IOXIC LIVER HYPERPLASIA OVARY CYST 91 TSA C 00 3 KIDNEY NEPHROSIS TOXIC 91 TSA C 00 4 KIDNEY NEPHROSIS TOXIC UTERUS RETENTION FLUID 91 TSA C 00 5 KIDNEY NEPHROSIS TOXIC ENDOMETRIUM HYPERPLASIA CYSTIC 91 TSA C 00 6 LIVE R HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC 91 TSA C 00 7 KIDNEY NEPHROSIS TCXIC 91 TSA C 00 8 LUN G ALVEOLAR ADENOCARCINOMA KIDNEY NEPHROSIS TOXIC LIVER HEPATOCELLULAR CARCINOMA

Continued on next page Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

91 TSAC 011 KIDNEY NEPHROSIS 1CXIC

91 TSA.C 012 LIVER HEFATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC UTERUS RETENTION FLUID

91 TSAC 013 LIVE R HEPATOCELLULAR CARCINOMA KIDNEY NEPHRCSIS TOXIC OVARY CYST

91 TSAC 014 KIDNEY NEPHROSIS TCXIC ENDOMETRIUM INFLAMMATION

91 TSAC 015 LUNG ADENOMA KIDNEY NEPHROSIS TOXIC

91 TSAC 016 KIDNEY NEPHRCSIS TOXIC

91 TSAC 017 MESENTERIC LYMPH NODE ANGIECTASIS UTERUS RETENTION FLUID KIDNEY NEPHROSIS TOXIC

91 TSAC 018 CERVICAL LYMPH NODE MALIGNAN T LYMPHOMA KIDNEY NEPHRCSIS TOXIC

91 TSAC 019 KIDNEY NEPHROSIS TCXIC

91 TSAC 021 KIDNEY NEPHROSIS TCXIC UTERUS RETENTION FLUID

91 TSAC 022 LIVE R LYMPHOSARCOMA OVARY CYST KIDNEY LYMPHOSARCOMA UTERUS RETENTION FLUID SPLEEN LYMPHOSARCOMA KIDNEY NEPHROSIS TOXIC

91 TSAC 023 LUN G ADENOMA KIDNEY NEPHROSIS TCXIC LIVER HYPERPLASIA

91 TSAC 024 LIVE R HEPATOCELLULAR CARCINOMA BRONCHUS BRONCHIECTASIS KIDNEY NEPHROSIS TOXIC

91 TSAC 025 LUNG ALVEOLAR ADENOCARCINOMA KIDNEY NEPHROSIS TOXIC LIVER HEPATOCELLULAR CARCINOMA OVARY CYST

91 TSAC 026 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC

91 TSAC 027 KIDNEY NEPHROSIS TOXIC

Continued on next page Table XXXIIb (continued). Individual Pathology - Trichloroethylene-Treated Female Mice

High Dose Group

WEEKS ON DISP ANIMAL TUMORS OTHER PATHOLOGY STUDY CODE NUMBER TOPOGRAPHY MORPHOLOGY TOPOGRAPHY MORPHOLOGY

91 TSAC 028 KIDNEY NEPHROSIS TOXIC ENDOMETRIUM HYPERPLASIA CYSTIC OVARY CYST 91 TSAC 029 KIDNEY NEPHROSIS TOXIC OVARY CYST 91 TSAC 031 KIDNEY NEPHROSIS TOXIC UTERUS RETENTION fLUID 91 TSAC 032 KIDNEY NEPHROSIS TOXIC 91 TSAC 033 KIDNEY NEPHROSIS TOXIC 91 TSAC 03a LUNG ADENOMA KIDNEY NEPHROSIS TOXIC 91 TSAC 035 KIDNEY NEPHROSIS TOXIC 91 TSAC 041 LUNG ADENOMA KIDNEY NEPHROSIS TOXIC MESENTERIC LYMPHNODE LYMPHOSARCOMA SPLEEN LYMPHOSARCOMA CERVICAL LYMPHNODE LYMPHOSARCOMA LIVER HEPATOCELLULAR CARCINOMA 91 TSAC 042 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHRCSIS TOXIC 91 TSAC 043 KIDNEY NEPHROSIS TOXIC 91 TSAC 044 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHROSIS TOXIC UTERUS RETENTION FLUID 91 TSAC 045 KIDNEY NEPHROSIS TOXIC UTERUS RETENTION FLUID 91 TSAC 046 LIVER HEPATOCELLULAR CARCINOMA KIDNEY NEPHRCSIS TCXIC LUNG ADENOMA 91 TSAC 047 KIDNEY NEPHROSIS TOXIC MESENTERIC LYMPH NODE ANGIECTASIS STOMACH ACANTHOSIS STOMACH HYPERKERATOSIS OVARY CYST 91 TSAC 048 KIDNEY NEPHROSIS OVARY CYST

end of female mice—high dose

APPENDIX E: POSITIVE CONTROLS

177 Table XXXIIIa. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Rats

Individual Week of Number Death Diagnosis

001 82 Portal cirrhosis Cholangietasis Fatty 002 110 Bile duct proliferation 003 103 Advanced autolysis 004 82 Hepatocellular carcinoma 005 110 Portal cirrhosis Bile duct proliferation Fatty Pigment deposition 006 110 Peliosis Bile duct proliferation Fibrosis (around bile ducts) 007 13 Centrilobular necrosis Fatty 008 106 Portal cirrhosis Cholangiectasis Fatty Bile duct proliferation 009 65 Portal cirrhosis Fatty Bile duct proliferation 010 95 Cholangiectasis Fatty 011 102 Portal cirrhosis Fatty Bile duct proliferation 012 62 Portal cirrhosis Fatty Bile duct proliferation 013 73 Fatty (diffuse) 014 44 Periportal necrosis Degeneration Fatty Fibrosis 015 99 Periportal degeneration Fibrosis Bile duct proliferation 016 107 Portal cirrhosis Pigment deposition 017 66 Portal cirrhosis Bile duct proliferation Fatty Neoplastic nodule

178 Table XXXIIIa. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Rats (continued)

018 110 Portal cirrhosis Bile duct proliferation Fatty 019 94 Portal cirrhosis Bile duct proliferation Fatty 020 110 Bile duct proliferation Angiectasis Fatty Regenerative nodules 021 105 Portal cirrhosis Bile duct proliferation Fatty 022 110 Angiectasis Bile duct proliferation Fatty Fibrosis 023 106 Advanced autolysis Portal cirrhosis Fatty 024 110 None 025 107 Portal cirrhosis Bile duct proliferation Regenerative nodules 026 110 Regenerative nodules Fatty Angiectasis 027 63 Portal cirrhosis 028 75 Bile duct proliferation Fibrosis Cholangiectasis 029 110 Bile duct proliferation Fatty Fibrosis 030 90 Myelogenous leukemia 031 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 032 60 Portal cirrhosis Bile duct proliferation Fatty Organizing thrombus Hepatocellular carcinoma Regenerative nodules 033 73 Portal cirrhosis Fatty Bile duct proliferation

179 Table XXXIlla. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Rats (continued)

034 71 Portal cirrhosis Bile duct proliferation Fatty Regenerative nodules 035 71 Portal cirrhosis Bile duct proliferation Regenerative nodules 036 82 Portal cirrhosis Bile duct proliferation 037 110 Fatty 038 101 Portal cirrhosis Hepatic abscess 039 108 Portal cirrhosis Bile duct proliferation 040 109 Bile duct proliferation Fibrosis Fatty Regenerative nodules 041 58 Portal cirrhosis Bile duct proliferation 042 30 Portal cirrhosis Bile duct proliferation Fatty 043 110 Portal cirrhosis Bile duct proliferation Fatty Regenerative nodules 044 69 Portal cirrhosis Bile duct proliferation Fatty 045 110 Neoplastic nodule 046 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 047 109 Portal cirrhosis Bile duct proliferation 048 104 Portal cirrhosis Bile duct proliferation 049 36 Reticulum cell sarcoma (multicentric) 050 110 Portal cirrhosis Bile duct proliferation Foci of altered cells Regenerative nodules

180 Table XXXIIIb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Rats

Individual Week of Number Death Diagnosis

001 107 Portal cirrhosis Bile duct proliferation Fatty 002 110 Portal cirrhosis Bile duct proliferation 003 100 Advanced autolysis Portal cirrhosis Bile duct proliferation 004 97 Hepatocellular carcinoma 005 80 Regenerative nodules 006 96 Advanced autolysis Bile duct proliferation Portal cirrhosis 007 96 Portal cirrhosis Bile duct proliferation 008 109 Regenerative nodules 009 91 Portal cirrhosis Bile duct proliferation Hepatitis 010 77 Portal cirrhosis Bile duct proliferation 011 55 Fatty 012 58 Portal cirrhosis Fatty 013 64 Portal cirrhosis Bile duct proliferation 014 81 Advanced autolysis Regenerative nodules 015 78 Portal cirrhosis Bile duct proliferation Fatty 016 61 Portal cirrhosis Bile duct proliferation Fatty 017 110 Portal cirrhosis Bile duct proliferation 018 68 Portal cirrhosis Bile duct proliferation Fatty Neoplastic nodule

181 Table XXXlIlb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Rats (continued)

019 40 Portal cirrhosis Fatty 020 97 Portal cirrhosis Bile duct proliferation Cholangiectasis 021 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 022 110 Portal cirrhosis Bile duct proliferation 023 77 Portal cirrhosis Bile duct proliferation 024 55 Portal cirrhosis Fatty Bile duct proliferation 025 104 Lymphocytic leukemia 026 79 Hepatocellular carcinoma 027 90 Portal cirrhosis Bile duct proliferation 028 100 Portal cirrhosis Bile duct proliferation 029 97 Portal cirrhosis Bile duct proliferation Organizing thrombus 030 65 Portal cirrhosis Bile duct proliferation Fatty 031 43 Portal cirrhosis Fatty Bile duct proliferation 032 109 Advanced autolysis Portal cirrhosis Bile duct proliferation 033 98 Portal cirrhosis Bile duct proliferation 034 93 Portal cirrhosis Bile duct proliferation 035 88 Portal cirrhosis Fatty Bile duct proliferation Foci of altered cells Regenerative nodules 036 110 Portal cirrhosis Bile duct proliferation 037 90 Portal cirrhosis Bile duct proliferation

182 Table XXXIIIb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Rats (continued)

038 14 Fat deposition, diffuse 039 100 Portal cirrhosis Bile duct proliferation 040 83 Portal cirrhosis Bile duct proliferation 041 109 Advanced autolysis Portal cirrhosis Regenerative nodules 042 73 Portal cirrhosis Bile duct proliferation Fatty 043 77 Portal cirrhosis Bile duct proliferation Fatty 044 110 Portal cirrhosis Bile duct proliferation 045 107 Portal cirrhosis Bile duct proliferation 046 96 Portal cirrhosis Bile duct proliferation 047 98 Portal cirrhosis Bile duct proliferation 048 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 049 54 Portal cirrhosis Fatty 050 85 Portal cirrhosis Bile duct proliferation

183 Table XXXIIIc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Rats

Individual Week of Number Death Diagnosis

001 100 Portal cirrhosis Bile duct proliferation 002 110 Neoplastic nodule 003 88 Regenerative nodules Bile duct proliferation 004 110 Foci of altered cells 005 71 Regenerative nodules Fatty 006 96 Portal cirrhosis Bile duct proliferation 007 105 Portal cirrhosis Bile duct proliferation 008 61 Fatty Portal cirrhosis Bile duct proliferation 009 110 Portal cirrhosis Bile duct proliferation Fatty Foci of altered cells Regenerative nodules 010 110 Portal cirrhosis Regenerative nodules 011 101 Reticulum cell sarcoma 012 110 Portal cirrhosis Fatty 013 104 Portal cirrhosis Bile duct proliferation Fatty Neoplastic nodule 014 110 Hepatocellular carcinoma 015 86 Portal cirrhosis Fatty 016 107 Portal cirrhosis Bile duct proliferation 017 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 018 110 Portal cirrhosis Fatty Bile duct proliferation 019 57 Portal cirrhosis Fatty 020 110 Portal cirrhosis Regenerative nodules

184 Table XXXIIIc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Rats (continued)

021 94 Portal cirrhosis Bile duct proliferation Fatty 022 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 023 100 Portal cirrhosis Organizing thrombus Pigment deposition Bile duct proliferation 024 88 Portal cirrhosis Regenerative nodules 025 110 Portal cirrhosis Angiectasis Hepatocellular carcinoma Bile duct proliferation 026 110 Portal cirrhosis Foci of altered cells Regenerative nodules 027 76 Portal cirrhosis Fatty Bile duct proliferation Hepatitis Regenerative nodules 028 110 Fatty Bile duct proliferation Regenerative nodules 029 88 Portal cirrhosis Bile duct proliferation Fatty 030 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 031 41 Portal cirrhosis Bile duct proliferation 032 93 Advanced autolysis Portal cirrhosis Bile duct proliferation 033 13 Advanced autolysis 034 94 Portal cirrhosis Fatty 035 73 Portal cirrhosis Bile duct proliferation Fatty 036 91 Portal cirrhosis Bile duct proliferation Angiectasis

185 Table XXXIIIc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Rats (continued)

037 110 Portal cirrhosis Bile duct proliferation 038 79 Advanced autolysis Portal cirrhosis Fatty 039 104 Advanced autolysis Portal cirrhosis Bile duct proliferation Fatty 040 74 Portal cirrhosis Fatty Bile duct proliferation 041 12 Portal cirrhosis Fatty 042 104 Portal cirrhosis Bile duct proliferation Hepatocellular carcinoma 043 110 Portal cirrhosis Fatty Bile duct proliferation Regenerative nodules 044 110 Portal cirrhosis Fatty 045 110 Portal cirrhosis Fatty Bile duct proliferation 046 58 Portal cirrhosis Fatty 047 39 Portal cirrhosis Bile duct proliferation 048 48 Portal cirrhosis Fatty Neoplastic nodule Hepatocellular carcinoma 049 110 Bile duct proliferation Fatty 050 110 Portal cirrhosis Bile duct proliferation

186 Table XXXIIId. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Rats

Individual Week of Number Death Diagnosis

001 110 Portal cirrhosis Bile duct proliferation Fatty 002 110 Portal cirrhosis Fatty Regenerative nodules 003 16 Portal cirrhosis 004 71 Portal cirrhosis Fatty 005 66 Portal cirrhosis Fatty Bile duct proliferation 006 110 Portal cirrhosis Bile duct proliferation Fatty Regenerative nodules 007 38 Portal cirrhosis Fatty 008 103 Portal cirrhosis Bile duct proliferation 009 110 Portal cirrhosis Fatty 010 110 Portal cirrhosis Bile duct proliferation Fatty Regenerative nodules 011 110 Portal cirrhosis Bile duct proliferation Regenerative nodules 012 82 Advanced autolysis Portal cirrhosis Bile duct proliferation 013 110 Portal cirrhosis Bile duct proliferation 014 110 Portal cirrhosis Bile duct proliferation Neoplastic nodule 015 110 Neoplastic nodule 016 12 Portal cirrhosis Fatty Bile duct proliferation 017 1 Portal cirrhosis Angiectasis 018 48 Portal cirrhosis Bile duct proliferation

187 Table XXXIIId. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Rats (continued)

019 80 Portal cirrhosis Bile duct proliferation 020 62 Portal cirrhosis Bile duct proliferation 021 52 Portal cirrhosis Fatty Bile duct proliferation Regenerative nodules 022 13 Portal cirrhosis Fatty 023 4 Portal cirrhosis Fatty 024 30 Portal cirrhosis Fatty 025 19 Portal cirrhosis Fatty 026 69 Portal cirrhosis Bile duct proliferation Hepatitis 027 110 Portal cirrhosis Bile duct proliferation Neoplastic nodule 028 34 Portal cirrhosis Bile duct proliferation Fatty 029 76 Portal cirrhosis Foci of altered cells Regenerative nodules 030 34 Lost 031 80 Portal cirrhosis Bile duct proliferation Regenerative nodules 032 70 Portal cirrhosis Bile duct proliferation Fatty Regenerative nodules 033 4 Portal cirrhosis Fatty 034 110 Regenerative nodules 035 110 Regenerative nodules 036 33 Portal cirrhosis Bile duct proliferation 037 110 Regenerative nodules 038 99 Advanced autolysis Portal cirrhosis Bile duct proliferation 039 12 Portal cirrhosis (early) Fatty

188 Table XXXIIId. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Rats (continued)

040 95 Portal cirrhosis Bile duct proliferation 041 104 Hepatocellular carcinoma 042 9 Portal cirrhosis Fatty Bile duct proliferation 043 53 Fatty Sinusoidal ectasia 044 29 Portal cirrhosis Bile duct proliferation 045 15 Portal cirrhosis Bile duct proliferation 046 44 Portal cirrhosis Bile duct proliferation Fatty Regenerative nodules 047 1 Fatty 048 50 Portal cirrhosis Bile duct proliferation 049 3 Fatty 050 110 Portal cirrhosis Bile duct proliferation Regenerative nodules

189 Table XXXIVa. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Mice

Individual Week of Number Death Diagnosis

001 80 Hepatocellular carcinoma 002 74 Hepatocellular carcinoma 003 72 Hepatocellular carcinoma 004 72 Hepatocellular carcinoma 005 72 Hepatocellular carcinoma 006 66 Hepatocellular carcinoma 007 65 Hepatocellular carcinoma 008 63 Hepatocellular carcinoma 009 50 Hepatocellular carcinoma 010 48 Hepatocellular carcinoma 011 80 Hepatocellular carcinoma 012 75 Hepatocellular carcinoma 013 74 Hepatocellular carcinoma 014 74 Hepatocellular carcinoma 015 73 Hepatocellular carcinoma 016 67 Hepatocellular carcinoma 017 67 Hepatocellular carcinoma 018 65 Hepatocellular carcinoma 019 63 Hepatocellular carcinoma 020 55 Hepatocellular carcinoma 021 82 Hepatocellular carcinoma 022 81 Hepatocellular carcinoma 023 79 Hepatocellular carcinoma 024 76 Hepatocellular carcinoma 025 76 Hepatocellular carcinoma 026 72 Hepatocellular carcinoma 027 70 Hepatocellular carcinoma 028 64 Hepatocellular carcinoma 029 61 Hepatocellular carcinoma 030 60 Hepatocellular carcinoma 031 86 Hepatocellular carcinoma 032 82 Hepatocellular carcinoma 033 81 Hepatocellular carcinoma 034 80 Hepatocellular carcinoma 035 75 Hepatocellular carcinoma 036 74 Hepatocellular carcinoma 037 71 Hepatocellular carcinoma 038 64 Hepatocellular carcinoma 039 54 Hepatocellular carcinoma 040 42 Autolysis

190 Table XXXIVa. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Male Mice (continued)

041 84 Hepatocellular carcinoma 042 80 Hepatocellular carcinoma 043 77 Hepatocellular carcinoma 044 77 Hepatocellular carcinoma 045 75 Hepatocellular carcinoma 046 72 Hepatocellular carcinoma 047 72 Hepatocellular carcinoma 048 69 Hepatocellular carcinoma 049 64 Hepatocellular carcinoma 050 50 Hepatocellular carcinoma

191 Table XXXIVb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Mice

Individual Week of Number Death Diagnosis

001 75 Hepatocellular carcinom a 002 75 Hepatocellular carcinom a 003 75 Hepatocellular carcinom a 004 66 Hepatocellular carcinom a 005 63 Hepatocellular carcinom a 006 63 Hepatocellular carcinom a 007 60 Hepatocellular carcinom a 008 60 Hepatocellular carcinom a 009 55 Hepatocellular carcinom a 010 52 Hepatocellular carcinom a on 90 Hepatocellular carcinom a 012 79 Hepatocellular carcinom a 013 77 Hepatocellular carcinom a 014 74 Hepatocellular carcinom a 015 69 Hepatocellular carcinom a 016 64 Hepatocellular carcinom a 017 56 Hepatocellular carcinom a 018 53 Hepatocellular carcinom a 019 42 Hepatocellular carcinom a 020 30 Hepatocellular carcinom a 021 77 Hepatocellular carcinom a 022 74 Hepatocellular carcinom a 023 66 Hepatocellular carcinom a 024 65 Hepatocellular carcinom a 025 62 Hepatocellular carcinom a 026 56 Cannibalized 027 48 Hepatocellular carcinom a 028 48 Autolysis 0 29 48 Hepatocellular carcinom a 030 26 Hepatocellular carcinom a 031 74 Hepatocellular carcinom a 032 74 Hepatocellular carcinom a 033 70 Hepatocellular carcinom a 034 65 Hepatocellular carcinom a 035 63 Hepatocellular carcinom a 036 60 Hepatocellular carcinom a 037 51 Hepatocellular carcinom a 038 51 Hepatocellular carcinom a 039 47 Hepatocellular carcinom a 040 47 Hepatocellular carcinoma

192 Table XXXIVb. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Male Mice (continued)

041 73 Hepatocellular carcinoma 042 73 Hepatocellular carcinoma 043 71 Hepatocellular carcinoma 044 70 Hepatocellular carcinoma 045 70 Hepatocellular carcinoma 046 58 Hepatocellular carcinoma 047 56 Hepatocellular carcinoma 048 35 Hepatocellular carcinoma 049 26 Hepatocellular carcinoma 050 16 Portal cirrhosis Bile duct proliferation

193 Table XXXIVc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Mice

Individual Week of Number Death Diagnosis

001 68 Hepatocellular carcinoma Organizing thrombus 002 77 Hepatocellular carcinoma 003 74 Hepatocellular carcinoma 004 74 Hepatocellular carcinoma 005 70 Hepatocellular carcinoma 006 68 Hepatocellular carcinoma 007 62 Cannibalized 008 62 Hepatocellular carcinoma 009 58 Cannibalized 010 45 Autolysis on 80 Hepatocellular carcinoma 012 80 Hepatocellular carcinoma 013 79 Hepatocellular carcinoma 014 75 Hepatocellular carcinoma 015 70 Hepatocellular carcinoma 016 68 Hepatocellular carcinoma Organizing thrombus 017 65 Hepatocellular carcinoma 018 64 Hepatocellular carcinoma 019 36 Hepatocellular carcinoma 020 29 Autolysis 021 81 Hepatocellular carcinoma 022 80 Hepatocellular carcinoma 023 75 Hepatocellular carcinoma 024 70 Hepatocellular carcinoma 025 66 Hepatocellular carcinoma 026 55 Hepatocellular carcinoma 027 48 Hepatocellular carcinoma 028 46 Hepatocellular carcinoma 029 33 Hepatocellular carcinoma 030 11 Autolysis 031 86 Hepatocellular carcinoma 032 84 Hepatocellular carcinoma 033 79 Hepatocellular carcinoma 034 78 Hepatocellular carcinoma 035 75 Hepatocellular carcinoma 036 66 Autolysis 037 66 Hepatocellular carcinoma 038 61 Hepatocellular carcinoma 039 54 Hepatocellular carcinoma 040 30 Hepatocellular carcinoma

194 Table XXXIVc. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - Low Dose Female Mice (continued)

041 81 Hepatocellular carcinoma 042 79 Cannibalized 043 78 Cannibalized 044 72 Cannibalized 045 71 Hepatocellular carcinoma 046 71 Hepatocellular carcinoma 047 69 Hepatocellular carcinoma 048 64 Cannibalized 049 55 Hepatocellular carcinoma 050 16 Hepatocellular carcinoma

195 Table XXXIVd. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Mice

Individual Week of Number Death Diagnosis

001 92 Hepatocellular carcinoma 002 68 Hepatocellular carcinoma 003 61 Hepatocellular carcinoma 004 61 Hepatocellular carcinoma 005 58 Hepatocellular carcinoma 006 51 Hepatocellular carcinoma 007 34 Lost 008 19 Hepatocellular carcinoma 009 19 Cannibalized 010 14 Toxic hepatitis Cirrhosis Bile duct proliferation Fatty 011 74 Hepatocellular carcinoma 012 66 Hepatocellular carcinoma 013 63 Hepatocellular carcinoma 014 60 Hepatocellular carcinoma 015 57 Hepatocellular carcinoma 016 52 Cannibalized 017 51 Autolysis 018 47 Hepatocellular carcinoma 019 43 Hepatocellular carcinoma 020 42 Hepatocellular carcinoma 021 78 Hepatocellular carcinoma 022 73 Hepatocellular carcinoma 023 71 Hepatocellular carcinoma 024 70 Hepatocellular carcinoma 025 67 Hepatocellular carcinoma 026 67 Hepatocellular carcinoma 027 67 Hepatocellular carcinoma 028 58 Hepatocellular carcinoma 0 29 54 Hepatocellular carcinoma 030 13 Portal cirrhosis Bile duct proliferation 031 80 Hepatocellular carcinoma 032 74 Hepatocellular carcinoma 033 68 Hepatocellular carcinoma 034 59 Hepatocellular carcinoma 035 57 Hepatocellular carcinoma 036 57 Hepatocellular carcinoma 037 56 Hepatocellular carcinoma 038 41 Hepatocellular carcinoma 039 38 Hepatocellular carcinoma 040 35 Hepatocellular carcinoma

196 Table XXXIVd. Liver Histopathology for Positive Controls (Carbon Tetrachloride) - High Dose Female Mice (continued)

041 80 Hepatocellular carcinoma 042 78 Hepatocellular carcinoma 043 77 Hepatocellular carcinoma 044 73 Hepatocellular carcinoma 045 67 Hepatocellular carcinoma 046 64 Cannibalized 047 46 Hepatocellular carcinoma 048 43 Hepatocellular carcinoma 049 42 Hepatocellular carcinoma 050 36 Hepatocellular carcinoma

197 198

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